CN106053799A - Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper - Google Patents
Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper Download PDFInfo
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Abstract
The invention relates to gold-labeled test paper for rapid determination of the crab vitellin content. The gold-labeled test paper comprises a base plate, a sample pad, a colloidal-gold pad, a nitrocellulose membrane layer and a water-absorbent paper layer. The colloidal-gold pad contains a gold-labeled anti-vitellin antibody compound formed from colloidal gold and an anti-vitellin antibody. The nitrocellulose membrane layer is provided with a test line printed by an anti-vitellin polyclonal antibody solution and a quality control line printed by a goat anti-mouse IgG antibody solution. The gold-labeled test paper has advantages that 1, the gold-labeled test paper is simple and convenient to use, fast in detection and high in sensitivity, and is greatly improved in the degree of reliability and work efficiency compared with visual observation and experiential inference for consumption situation of vitellus substances; and 2, the gold-labeled test paper is used for rapid determination of the vitellin content of crab aquatic products, provides a visualized and reliable determination result, and is used for a standardized test of crab breeding technology. The success rate of artificial crab breeding is increased, and basis for sustainable development of economic crab culture industry is provided.
Description
Technical field
The present invention relates to vitellin detection technique field, specifically, be a kind of quickly mensuration Eriocheir sinensis class yolk phosphorus egg
The gold test strip of Bai Hanliang and application thereof.
Background technology
Economic crab is the important component part of China's aquatic products, adds up through China Fisheries yearbook, and to 2014, China was each
Class economic crab apparent consumption reaches 600,000 tons, and main economic crab includes Eriocheir sinensis, portunus trytuberculatus, intends cave
Mud crab, Portunus pelagicus etc., for adapting to the great demand in market, China progressively starts various economy from last century the eighties
The artificial cultivation of Eriocheir sinensis class, along with the continuous expansion of economic crab cultivation scale, the subject matter of restriction cultivation development is high-quality Seedling
Plant rare.Owing to Eriocheir sinensis class is different with Fish, will be through shell for several times after hatching, metamorphosis just can become " Eriocheir sinensis truly
Seedling ", this complex characteristics causes current Eriocheir sinensis class nursery level the most on the low side, and parent's gonad development is bad, egg load is low, embryo sends out
Educating that mortality rate is high, germling incubation rate is low etc. all causes the under-supply of high-quality seed.Additionally, the seedling-raising technique of poor efficiency causes often
Needing to capture substantial amounts of parent from wild resource year or natural seeding, wild fry could meet Production requirement, this necessarily gives economic crab
Natural resources protection bring huge pressure, therefore improve the success rate of Eriocheir sinensis class artificial breeding, be economic crab aquaculture industry
The basis of sustainable development.
Shell-fish is respectively formed yolk sac structure in embryo development procedure, and the Yolk in yolk sac is that whole embryo sends out
Unique source of nutrition during educating, after hatching, still has part Yolk in yolk sac, provide energy for Newly hatchled
Amount, treats that Yolk is exhausted, and yolk sac specialization is hepatopancrease, and germling just starts to ingest from the external world acquisition nutrition.Due to first
Still by the characteristic of self Yolk energy supply a period of time after this hatching of shell animal, bring the biggest difficulty to nursery work
Topic, bait throwing in well-timed after zoea hatching, it is to ensure that one of successful key factor of nursery.Too early bait throwing in, germling
Not ingesting, bait affects water quality, too late bait throwing in water, and ensuing metamorphosis, already at starvation, can be sent out by germling
Educating and cause irreversible injury, optimal bait throwing in is that Yolk is just exhausted in germling body opportunity, starts initial feeding.
In current Eriocheir sinensis class nursery produces, judge by experience when starting bait throwing in, the Newly hatchled of difference crustaceans always
Entrained Yolk also differs, and therefore, the optimum bait throwing in of different crustacean larvae all differs opportunity.Even if
Being same species, owing to parent's nourishment is different, the difference of the environmental factorss such as water temperature and quality, when all can affect the suitableeest bait throwing in
Machine.As can be seen here, micro-judgment the suitableeest bait throwing in not only accuracy rate on opportunity is low the most artificially, and cannot quantify, and hinders and educates
The standardized and popularized of Seedling technology, needs badly the most at present and a kind of can the most effectively determine the detection hands of Yolk content in germling body
Section breaks through this bottleneck.
Summary of the invention
It is an object of the invention to for deficiency of the prior art, it is provided that a kind of quickly mensuration Eriocheir sinensis class vitellin content
Gold test strip.
Another purpose of the present invention is to provide the purposes of gold test strip described above.
For achieving the above object, the present invention adopts the technical scheme that:
The gold test strip of a kind of quick mensuration Eriocheir sinensis class vitellin content, including base plate, sample pad, gold size pad, nitric acid
Fibrous membrane layer and absorbent paper layer, described base plate covers sample pad, gold size pad, nitrocellulose membrane layer and absorbent paper layer, institute successively
State and be combined the gold mark anti-vitellin antibody complex formed, institute with anti-vitellin monoclonal antibody containing gold colloidal on gold size pad
State nitrocellulose membrane layer be provided with anti-vitellin Anti-TNF-α liquid solution print p-wire (T line) and sheep anti-mouse igg resist
The nature controlling line (C line) that liquid solution is printed.
Further, it is as follows that described gold marks anti-vitellin antibody complex preparation method: takes colloidal gold solution, adds
0.2mol/L K2CO3Solution, K2CO3Addition is 7 μ L/mL, is subsequently adding the anti-vitellin monoclonal antibody dialysed, monoclonal antibody
Addition is 10 μ g/mL, adds the BSA mixing of final concentration of 1% after overnight stablizing.
Further, in described nature controlling line, antibody is coated concentration is 1mg/mL.
Further, in described p-wire, antibody is coated concentration is 1mg/mL.
Further, described gold test strip is limited to 1ug/ml for the detection of Eriocheir sinensis vitellin.
Further, described gold size pad is made up of glass fibre and the golden labeling antibody being fixed thereon, and described sample pad is
Water absorption glass fibre.
For realizing above-mentioned second purpose, the present invention adopts the technical scheme that:
The application in quickly detection Eriocheir sinensis class aquatic products vitellin content of arbitrary described gold test strip.
The present invention differentiates in sample whether contain vitellin based on colloidal gold antibody competitive binding principle.Described
Antibody is to be mouse monoclonal antibody prepared by antigen by purification vitellin in Eriocheir sinensis female parent ovary, and described gold colloidal resists
The carrier of body is nitrocellulose filter, and quality control region and test section on described carrier are coated rabbit anti-mouse igg respectively.By to be detected
Dripping on reagent paper after germling homogenate, vitellin as internal in it is not yet exhausted, and surveys at p-wire C and nature controlling line T equal
Red stripes occurs, if its internal vitellin has been exhausted, then without band at p-wire, only has at nature controlling line
Band.
The invention has the advantages that:
1, the gold test strip of the present invention is easy to use, and detection is quickly, highly sensitive, and relatively range estimation and experience infer Yolk
Expenditure Levels reliability and work efficiency are all greatly improved.
2, the optimum bait throwing in of different crustacean larvae all differs opportunity, even same species, due to parent
Nourishment is different, the difference of the environmental factorss such as water temperature and quality, all can affect the suitableeest bait throwing in opportunity；The gold test strip inspection of the present invention
Surveying for quickly detection Eriocheir sinensis class aquatic products vitellin content, result judges intuitive and reliable, for the mark of Eriocheir sinensis class seedling-raising technique
Standardization is tested, and improves the success rate of Eriocheir sinensis class artificial breeding, for the basis of economic crab aquaculture industry sustainable development.
Accompanying drawing explanation
Accompanying drawing 1 is rabbit multi-resistance wb testing result.
Accompanying drawing 2 is rabbit multi-resistance elisa testing result.
Accompanying drawing 3 is the actually used testing result of gold test strip of the present invention.
Accompanying drawing 4 is the gold test strip structural representation of the present invention.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate this
Bright rather than limit the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention records, art technology
The present invention can be made various changes or modifications by personnel, and these equivalent form of values fall within the application appended claims equally and limited
Fixed scope.
The preparation of embodiment 1 gold test strip and detection
1 vitellin extracts
Take the mature ovarian of 2.0g Eriocheir sinensis, add the homogenate buffer ice bath homogenate of 5ml pre-cooling, then will homogenate
Liquid is centrifugal (centrifugal force=12000 × g) 30 minutes at 4 DEG C, take purple supernatant 2-3ml, and add isopyknic saturated sulfur
Acid ammonium solution, is centrifuged (centrifugal force=12000 × g) 30 minutes at 4 DEG C after ice bath, abandons supernatant, adds 2ml in centrifuge tube
PBS dissolution precipitation, the most in triplicate, finally precipitate is dissolved in the PBS of 1ml, treats gel mistake
Filtering layer is analysed.Use purification instrument (model: BioLogic DouFlowTM, the U.S. Bole public affairs of the full-automatic proteins and peptides of Bole
Department produces) ovary extract is carried out isolated and purified.Wherein gel column volume be 120ml (diameter × highly=2crn × 40cm,
Shanghai Sha Mei biotechnology Development Co., Ltd produces), load 30ml gel (model Sephaeryl S-300 Sweden
Pharmacia company produces), before purification initially with PBS solution balanced gel post, flow velocity 3mL/min, balance 3 hours.With note
Emitter loading lml Vn extracting solution (protein concentration is about lmg/ml), flow velocity is 0.6ml, detects under the conditions of 280nm and 470nm
The absorbance (OD) of eluent, when there is protein component, every lmL collects once.Eluent saves backup in-70 DEG C.
2 protein purifications
Acrylamide monomer reservoir: 14.55g acrylamide adds 0.45g N, N'-methylene diacrylamide, first uses 40mL
Distilled water stirring and dissolving, until solution becomes transparent diluter to 50mL with distilled water, filter.Save backup with brown bottle 4 DEG C.
Concentrate glue buffer reservoir (0.5mol/L Tris-HCl, pH6.8): 3.03gTris is dissolved in 40mL distilled water
In, adjust pH6.8 with 4mol/L hydrochloric acid.Dilute to 50mL with distilled water again.Be saved in 4 DEG C standby.
Separation gel buffer reservoir (1.5mol/L Tris-HCl, pH8.9): 18.16gTris is dissolved in 80mL distilled water
In, adjust pH8.9 with 4mol/L hydrochloric acid.Again with distilled water dilute to 100mL, be saved in 4 DEG C standby.
10% (AP) Ammonium persulfate.: 0.1g Ammonium persulfate. dissolves in 1.0mL distilled water, uses front Fresh.
Electrode buffer (0.025mol/L Tris, 0.2mol/L glycine, pH8.3): 15.14gTris adds 72.07g
Glycine, is diluted to 5L with distilled water.Can be stored at room temperature one month.
It is sweet plus 1mL 87% that sample buffer (0.1mol/L Tris-HCl, pH6.8): 2ml concentrates glue buffer reservoir
Oil, 0.1mg bromophenol blue, be diluted to 10mL with distilled water, can preserve 6 months at-20 DEG C.
Separation gel formula: distilled water: 6.6ml, 30% acrylamide solution: 8.0ml, 1.5mol/L Tris (pH8.8):
5.0ml, 10%AP (W/V): 200ul, TEMED:15ul.
Concentration glue formula: distilled water: 6.8ml, 30% acrylamide solution: 1.7ml, 1mol/LTris (pH6.8):
1.25ml, 10% Ammonium persulfate. (W/V): 100ul, TEMED:10ul.
By glass plate, rubber cushion, comb distilled water wash clean, use cotton ball soaked in alcohol wiping, electrophoresis tank is installed, preparation point
From glue (12%) and concentration glue (5%).Ammonium persulfate. and TEMED are eventually adding, and add post polymerization and i.e. start, and mixing immediately is poured into
Between two pieces of glass plates.Separation gel is poured between two pieces of glass plates, leaves applicable height, makes loading wells front end have from separation gel
The distance of about 2.5cm, is slowly added into the high distilled water of about 0.5cm at glue top, after glue to be separated polymerization completely, and upper strata of inclining
Distilled water, with distilled water clean gel top layer, with absorbent paper suck remnants water droplet.Pour concentration glue into glass plate interlayer,
Plug comb, after glue to be concentrated polymerization completely, take out comb, clean loading wells with distilled water immediately, add electrode buffer.
Eriocheir sinensis vitellin eluent is mixed with sample-loading buffer 4:1 after PBS is diluted to concentration 20ug/ul
Close, click and enter bottom loading wells with microsyringe, 200 volts of electrophoresis.When bromophenol blue arrives separation gel, voltage changes 250 volts into, continues
Continuous electrophoresis arrives bottom gel to bromophenol blue.Gel is peeled, is immersed in the substrate solution of 100ml, dye 1 hour, treat adhesive tape
Take a picture immediately after colour developing.Then gel is dyeed.Eriocheir sinensis vitellin is made up of two subunits, brings bar into
Albumen is reclaimed in row rubber tapping.
The preparation of 3 polyclonal antibodies
Protein solution will be reclaimed with isopyknic Freund adjuvant, YOULONG adjuvant mixing and emulsifying uniformly, become Water-In-Oil shape
State, in case immune rabbit.Immunization strategy: 2 new zealand white rabbits of immunity, subcutaneous inoculation 3 times, is spaced one month, after warp
ELISA detects, antiserum titre > 1:50000.Take blood: auricular vein booster immunization, after booster immunization two weeks, gather Sanguis Leporis seu oryctolagi
(all gathering).Polyclonal antibody purification: Sanguis Leporis seu oryctolagi is centrifuged 15min (4000rpm, room temperature), takes upper serum, under stirring at 4 DEG C
Dropwise it is slowly added to saturated ammonium sulfate and stirs 30min to semi-saturation, continuation, be centrifuged 30min (13000rpm, 4 DEG C), abandon supernatant；
Precipitation is dissolved in appropriate PBS (0.01M, pH7.4)；Under 4 DEG C of stirrings, dropwise it is slowly added to saturated ammonium sulfate to 33%, continues stirring
30min, centrifugal 30min (13000rpm, 4 DEG C), abandon supernatant；Precipitation is dissolved in appropriate PBS (0.01M, pH7.4), dialyses for 4 DEG C
At night, measuring antibody content ,-20 DEG C frozen standby.Continue after ammonium sulfate precipitation to use Protein A pillar to be purified, new post
Son first crosses post with 5ml ultra-pure water, then balances purification pillar with 5ml 0.4M PB buffer (pH 7.0)；Antibody crosses post, during
Require slow post excessively, be preferably combined on binding site in the hope of antibody protein；Continue 10ml0.4M PB buffer (pH 7.0)
Balance purification pillar；Antibody on 5ml 0.1M glycine-HCI buffer (pH 3.0) elution of bound site, and add 1M
Tris-HCl (pH 8.0) neutralizes glycine, makes pH remain the neutrality that applicable antibody preserves.
The preparation of 4 monoclonal antibodies
The albumen of expression and purification is uniform with isopyknic Freund adjuvant mixing and emulsifying, become Water-In-Oil state, in case immune
Mice.Immunization strategy: by 4 mices of protein immunization, subcutaneous inoculation 3 times, be spaced 4 weeks, after detect through ELISA, antiserum drips
Degree > 1:32000.Cell merges: last immune two weeks after, lumbar injection antigen carries out booster immunization, carries out thin after three days
Born of the same parents are merged.The neck that broken by mice is put to death, 70% soak with ethanol 30min sterilization, cuts off abdominal cavity at super-clean bench, takes out spleen, grind, mistake
80 eye mesh screens, obtain splenocyte, add SP2/0 myeloma cell, carry out cell fusion under the effect of PEG4000.Merge sieve
Choosing: spread the cell merged into 96 orifice plates, cultivate with HAT culture fluid, change liquid after three days, uses HT culture fluid instead and cultivates.
After 10 days, take cells and supernatant and detect.Cloning with build strain: use limiting dilution assay that positive hole is carried out cloning,
Detect after 10 days, positive colony is continued limiting dilution assay and carries out cloning, until the clone obtained is positive, sun can be set up
Sexual cell strain, obtains positive cell strain 27 strain altogether.Amplification culture: the monoclonal cell amplification culture of strain will be built, and carry out frozen.
Prepared by ascites: carry and inject mineral oil at mouse peritoneal the last week, and a number of cell infusion is entered mouse peritoneal,
Within about 10 days, collecting ascites, 4000rpm is centrifuged, and obtains supernatant and is monoclonal antibody ascites.Monoclonal antibody-purified: ascites is centrifuged
15min (4000rpm, room temperature), takes supernatant, is dropwise slowly added to saturated ammonium sulfate to semi-saturation, continuation stirring under 4 DEG C of stirrings
30min, centrifugal 30min (13000rpm, 4 DEG C), abandon supernatant；Precipitation is dissolved in appropriate PBS (0.01M, pH7.4)；Under stirring at 4 DEG C
Dropwise it is slowly added to saturated ammonium sulfate to 33%, continues stirring 30min, centrifugal 30min (13000rpm, 4 DEG C), abandon supernatant；Heavy
Shallow lake is dissolved in appropriate PBS (0.01M, pH7.4), 4 DEG C of dialysed overnight, measures antibody content, and-20 DEG C frozen standby.Ammonium sulfate precipitation
Rear continuation uses Protein G pillar to be purified, and new pillar first crosses post with 5ml ultra-pure water, then with 5ml 0.4M PB buffer
(pH 7.0) balances purification pillar；Antibody crosses post, during require slow cross post, be preferably combined in combination in the hope of antibody protein
On site；Continue 10ml 0.4M PB buffer (pH 7.0) balance purification pillar；5ml 0.1M glycine-HCI buffer
Antibody on (pH 2.7) elution of bound site, and add 1M Tris-HCl (pH 8.0) neutralization glycine, make pH remain suitable
Close the neutrality that antibody preserves.
5 polyclonal antibody titrations
Use the titer of sample direct coating ELISA inspection rabbit polyclonal antibody.First by Vn (the yolk phosphorus egg of purification
In vain) become 1 μ g/ml with being coated buffer solution, be then coated with 100 μ l/ holes；Vn antiserum after purification is pressed 1:200,
1:1000,1:5000,1:10000,1:20000,1:60000,1:240000 are diluted with the BSA of 1%, and every hole is loaded 100 μ
L, finally measures OD450 reading in microplate reader.Use Checkerboard titration method determine antibody and antigen the suitableeest working concentration (Fig. 1-
2), antigen Vn is coated the Concentraton gradient of liquid is 8.05,16.09,32.19,64.38,128.75,257.5,515 and 1030ng/
Ml, each concentration is laterally loaded 9 holes, arranges string simultaneously and is coated buffer as negative control, interpolation without Vn's；Vn resists
Body extension rate is divided into 1:1000,1:4000,1:8000,1:16000,1:32000,1:64000, antibody to dilute 1.6-6.4 ten thousand
During multiple, OD450 reading differs only by 0.19.When antibody dilutes 64000 times, OD450 reading is still up to 1.54, and this explanation should
The titer that antibody specific is higher, the ELISA that still can be used for Eriocheir sinensis Vn content in dilution for more than 20,000 times measures.
6 antibody conjugates screenings
By dilute according to 1:100 times for the carbonate buffer solution (being coated liquid, lower same) of 27 strains Mus monoclonal antibody pH9.6 after purification
Releasing, 100 μ L/ holes, 4 DEG C overnight.Washing liquid is cleaned and is coated plate 3 times, and each washing liquid consumption 300uL, the time is 1min, 10% calf blood
Clear closing (diluting according to 1:9 times with the carbonate buffer solution of PH9.6), every hole 150 μ L, constant temperature 37 DEG C is closed 3 hours.Add dense
Degree gradient is 1000,100,10, the vg protein solution of 0ppb, 100 μ L/ holes, 25 DEG C, incubation 45min.1:1000 is pressed with diluent
Dilution proportion enzyme labelled antibody, fully mixes, every hole 100 μ l, 25 DEG C, incubation 45min.Washing liquid is cleaned and is coated plate 3 times, each washing liquid
Consumption 300 μ L, the time is 1min, and every hole adds TMB 100 μ L, 25 DEG C of reaction 15min.Add 2M H2SO4, 100 μ L/ holes, 450nm
Read OD (absorbance) value.In 27 strain monoclonal antibodies, major part monoclonal antibody is fine with rabbit multi-resistance pairing effect, and sensitivity can reach 1ppb
Above.
The preparation of 7 gold medal labeling antibody complex
(1) configuration of colloidal gold solution: take 0.01% aqueous solution of chloraurate 100ml and be heated to boiling, add while stirring
1% trisodium citrate aqueous solution 1.8ml, continues to boil cooling after 10min is cerise to solution.Load in bag filter after cooling
Ultra-pure water (1:5000) is dialysed three times, finally the gold colloidal dialysed is transferred in the vial of clean band spiral cover, 4
Preserve in the environment of DEG C lucifuge.
(2) configuration of gold labeling antibody
The determination of labelling pH: take the centrifuge tube of 6 1.5mL, carries out 1-6 numbering to it, and accurate in each centrifuge tube
Add 1.0mL colloidal gold solution.The most each pipe is separately added into 0 μ L, 1 μ L, 3 μ L, 5 μ L, 7 μ L, the 0.2mol/L of 9 μ L
K2CO3, after shaking up, in each pipe, add 15 μ g monoclonal antibodies respectively, after shaking up, stand 40min, the most often pipe is separately added into 100 μ L
10%NaCl, stands and observes the interior gold colloidal cosmetic variation of often pipe, if color occurs substantially to become blue, the Guan Zhongwei producing precipitation does not conforms to
Suitable pH, and color change is unconspicuous for suitable pH.Colloidal gold solution is centrifuged 10min, takes supernatant and measures the OD of 520nm
Value, the absorbance of No. 5 pipes is the highest, and 0.2mol/L K is described2CO3For the suitableeest addition when addition is 7 μ L/mL.
The determination of optimum mark concentration: take the centrifuge tube of 7 1.5mL, carries out 1～9 numberings to it, and at each centrifuge tube
In accurately add 1.0mL colloidal gold solution.The most each pipe is separately added into the 0.2mol/L K of 7.0 μ L2CO3, after shaking up respectively
In each pipe, add 0 μ g, 2 μ g, 4 μ g, 6 μ g, 8 μ g, 10 μ g, 12 μ g, 14 μ g, 16 μ g monoclonal antibodies, after shaking up, stand 40min,
The NaCl solution of the 10% of rear addition 100 μ L, in each pipe, stands and watches the change of gold colloidal outward appearance in each group of pipe.If color
Occur significant change to become indigo plant, produce Guan Zhongwei inappropriate antibody addition of precipitation, and color still can keep red change
Unconspicuous for suitable antibody addition, and the most non-discoloring for minimum mark amount.Colloidal gold solution is centrifuged 10min, takes
Supernatant measures the OD value of 520nm, and 1-5 pipe occurs that color changes, and the absorbance of No. 6 pipes is the highest, illustrates in colloidal gold solution
For the suitableeest antibody addition when antibody addition is 10 μ g/mL.
The configuration of gold labeling antibody: take 1L colloidal gold solution, adds 0.2mol/L K2CO3Solution, K2CO3The addition of solution
It is 7 μ L/mL.Being subsequently adding the anti-vitellin monoclonal antibody dialysed, the addition of monoclonal antibody is 10 μ g/mL, adds after overnight stablizing
Enter the BSA mixing of final concentration of 1%.
(3) purification of gold colloidal albumen: the colloidal gold labeled monoclonal antibody complex prepared is centrifuged at 900rpm/min
15min, careful sucking-off supernatant, precipitates and answers with containing the sucrose of 5% and the 0.002M borate buffer solution of 0.05%Tween-20
Molten.Centrifuge washing twice, is finally concentrated into complex the 1/10 of original volume, saves backup in 4 DEG C.
The preparation of 8 colloidal gold strips
Refer to Fig. 4, Fig. 4 is the structural representation measuring vitellin content gold test strip.The labelling related in Fig. 4
As follows: 1. base plate, 2. sample pad, 3. gold size pad, 4. nitrocellulose membrane layer, 5. absorbent paper layer.This reagent paper is provided with ground base plate 1, the end
Sample pad 2, gold size pad 3, nitrocellulose membrane layer 4 and absorbent paper layer 5 is covered successively on plate.Described base plate 1 is PVC base plate, sample
The material of pad 2 is glass fibre.The making of gold size pad: the gold mark monoclonal antibody body point gold mark film machine that above-mentioned steps 7 is prepared
Being sprayed on uniformly on the gold mark pad processed, point sample amount is 2 μ L/cm, dries 24 hours in 37 DEG C, standby.Draw film: choose PALL
NC film 170 draw p-wire (T line) by how anti-zoned for rabbit that concentration is 1.0mg/mL with 0.7 μ L/cm with some gold mark (film) machine；By dense
The sheep anti mouse two that degree is 1.0mg/mL resists draws nature controlling line (C line) with 0.7 μ L/cm, dries 24 hours in 37 DEG C, standby.
9 is actually used
Purification vitellin is tested, and Eriocheir sinensis vitellin good for purification is configured to 0.5,1,10,20 μ g/
The solution of mL, puts in 1.5ml centrifuge tube, and sets the solution without vitellin as blank, by the reagent paper rule according to
Arrow is inserted in centrifuge tube, reads result after 10 minutes, and detection is limited to 1ug/ml (Fig. 3).
Eriocheir sinensis zoea vitellin method for quick, collects Eriocheir sinensis zoea 80-100
Only left and right, puts in 1.5ml centrifuge tube, addition 1ml homogenate buffer (1M Tris HCl 10ml, NaCl 2.925g,
EDTA0.05g, 100mM PMSF 0.5ml, adds distilled water and is settled to 500ml), grind in centrifuge tube 5 minutes with grinding rod,
Static 15 minutes, 12000 leave the heart 5 minutes subsequently, take supernatant.Test strips is inserted supernatant, after 10 minutes, reads result,
As two lines occurs, then show zoea still contains the vitellin not digested.
Embodiment 2T line and the optimal coated screening of C line
When making NC film detection layers, the antibody of detection line and nature controlling line is coated concentration and directly affects the colour developing effect of test strips
Really.As nature controlling line antibody is coated excessive concentration, C line color is the deepest, and the too low colour developing of concentration is unintelligible, does not has nature controlling line effect；
The antibody of detection line is coated excessive concentration not only affects the color developing effect of nature controlling line, and false positive easily occurs, wastes antibody；
Concentration too low T line developed the color shallow, affected the interpretation of result.The present invention is coated concentration to the antibody of nature controlling line and detection line and sieves
Choosing, experimental technique is same as in Example 1, T line on NC film and C line be coated 0.1 respectively, 0.5,1,2mg/mL antibody, shape
Becoming 16 different combinations, be assembled into test strips, selecting Eriocheir sinensis vitellin concentration is that 20 μ g/mL test,
Observe nature controlling line and the colour developing of detection line, determine the optimum concentration of coated antibody.The antibody of variable concentrations is coated NC film detection layers
Result as shown in table 1, when detecting line coated antibody concentration and being 0.1mg/mL, detection line color is shallower, and naked eyes just can be differentiated；
When detecting line coated antibody concentration and being 0.5mg/mL, detection line has colour developing but to compare nature controlling line color developing effect more weak；Work as detection
When line coated antibody concentration is 1mg/mL, detection line and nature controlling line color are all clear substantially；When detection line is coated sky concentration degree it is
During 3mg/mL, retain more golden labeling antibody owing to detection line concentration is higher and make nature controlling line colour developing weaken.From reagent paper cost and
From the standpoint of detection colour developing result two, the final antibody selecting control line and detection line is coated concentration and is respectively 1mg/mL and 1mg/mL
For optimum concentration.
Table 1 T line and the optimal coated concentration combination of C line
Note: detection line, nature controlling line: +++ Strong positive signals；+++ positive signal；+ weak positive model；-no positive signal.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of without departing from the inventive method, it is also possible to makes some improvement and supplements, and these improve and supplement and also should be regarded as
Protection scope of the present invention.
Claims (7)
1. a gold test strip for quick mensuration Eriocheir sinensis class vitellin content, including base plate, sample pad, gold size pad, nitric acid fibre
Dimension film layer and absorbent paper layer, described base plate covers sample pad, gold size pad, nitrocellulose membrane layer and absorbent paper layer successively, and it is special
Levy and be, described gold size pad is combined with anti-vitellin monoclonal antibody containing gold colloidal the gold mark anti-vitellin antibody formed
Complex, described nitrocellulose membrane layer is provided with p-wire (T line) and the sheep that anti-vitellin Anti-TNF-α liquid solution is printed
The nature controlling line (C line) that dynamics solution is printed.
The most quickly measure the gold test strip of Eriocheir sinensis class vitellin content, it is characterised in that described gold
Mark anti-vitellin antibody complex preparation method as follows: take colloidal gold solution, add 0.2mol/L K2CO3Solution, K2CO3
Addition is 7 μ L/mL, is subsequently adding the anti-vitellin monoclonal antibody dialysed, and the addition of monoclonal antibody is 10 μ g/mL, the most surely
The BSA mixing of final concentration of 1% is added after Ding.
The most quickly measure the gold test strip of Eriocheir sinensis class vitellin content, it is characterised in that described matter
In control line, antibody is coated concentration is 1mg/mL.
The most quickly measure the gold test strip of Eriocheir sinensis class vitellin content, it is characterised in that described survey
In examination line, antibody is coated concentration is 1mg/mL.
The most quickly measure the gold test strip of Eriocheir sinensis class vitellin content, it is characterised in that described gold
Mark reagent paper is limited to 1ug/ml for the detection of Eriocheir sinensis vitellin.
The most quickly measure the gold test strip of Eriocheir sinensis class vitellin content, it is characterised in that described
Gold size pad is made up of glass fibre and the golden labeling antibody being fixed thereon, and described sample pad is water absorption glass fibre.
7. the arbitrary described gold test strip of claim 1-6 answering in quickly detection Eriocheir sinensis class aquatic products vitellin content
With.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610629061.3A CN106053799A (en) | 2016-08-03 | 2016-08-03 | Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610629061.3A CN106053799A (en) | 2016-08-03 | 2016-08-03 | Gold-labeled test paper for rapid determination of crab vitellin content and application of colloidal-gold test paper |
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|---|---|---|---|---|
| CN1763090A (en) * | 2005-10-11 | 2006-04-26 | 中国水产科学研究院黑龙江水产研究所 | Sturgeon family fish ovovitellin preparation method and uses |
| CN101402672A (en) * | 2008-11-06 | 2009-04-08 | 中国石油大学(华东) | Method for extracting water-soluble protein component of yolk |
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| CN1763090A (en) * | 2005-10-11 | 2006-04-26 | 中国水产科学研究院黑龙江水产研究所 | Sturgeon family fish ovovitellin preparation method and uses |
| CN101402672A (en) * | 2008-11-06 | 2009-04-08 | 中国石油大学(华东) | Method for extracting water-soluble protein component of yolk |
| CN102440202A (en) * | 2011-09-29 | 2012-05-09 | 中国水产科学研究院黑龙江水产研究所 | Screening method for roes of fishes in Acipenseridae |
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