CN106336458A - Vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and preparation method thereof - Google Patents
Vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a vibrio parahaemolyticus colloidal gold-yolk antibody rapid detection test paper and a preparation method thereof; with vibrio parahaemolyticus as a research object, yolk antibodies are prepared with an outer membrane protein and hemolysin as immunogens, and high-purity ompK yolk antibody and a high-purity TDH yolk antibody are obtained successively through a water extraction method, a saturated ammonium sulfate precipitation method and a SephadexG-75 dextran gel chromatography extraction. With the vibrio parahaemolyticus THD yolk antibody as a capture antibody and the vibrio parahaemolyticus ompK yolk antibody as a detection antibody, the vibrio parahaemolyticus rapid detection test strip is developed; the rapid test strip has the advantages of sensitiveness and convenience in use, can be widely applied in early rapid testing of aquaculture animal diseases, and has a good application prospect.
Description
Technical field
The present invention relates to the technical field to the detection of aquatic animal pathogenic conditions aspect in aquaculture, especially a kind of
Quick detection test paper of vibrio parahaemolytious and preparation method thereof.
Background technology
In recent years, with the continuous expansion of sea-farming scale, disease frequently occurs, to our province or even national sea-farming
Cause huge threat with development, become one of Main Bottleneck that restriction aquaculture develops in a healthy way, wherein especially with vibriosises
For serious.On the other hand, in coastal area, the cause of disease distribution of microbes alimentary toxicosis there occurs significant changes, in marine product
There is the vibrio that many can cause human diseasess, wherein by vibrio parahaemolytious (vibrio parahaemolyticus), molten algae
The food that vibrio (v.alginolyticus), vibrio cholera (v.cholera) and Vibrio vulnificus (v.vulnificus) etc. cause
Poisoning is especially notable.And the alimentary toxicosis that vibrio parahaemolytious cause, it has been in microbes aquatic products alimentary toxicosis first
Position.It has been demonstrated that vibrio parahaemolytious are to cause marine cultured animal principal causative vibrio, seriously threaten Carnis Pseudosciaenae
(pseudosciaena crocea), Paralichthys olivaceuss (paralichthysolivaceus), snapper (pagrosomus major), stone
Speckle fish (epinephelus sp.), Penaeus vannamei (penaeus vannamei), Conch Meretricis seu Cyclinae (meretrix mertrix) etc. are big
Most marine cultured animals, the industry crisis that particularly nearly 2 years Penaeus vannamei industries run into, is all to cause a disease because infection is high
The acute Hepatopancreatic necrosis disease that property vibrio parahaemolytious cause is caused.Chinese scholars are also carried out to the virulence factor of morbid vibrio
Isolate and purify with Study on Pathogenicity it was recently reported that the major virulent factor of vibrio includes iron uptake system, thalline component (lipopolysaccharide
Lps and outer membrane protein ompk etc.), extracellular productses (hemolysin tdh and extracellular protease etc.).However, also lacking maturation at present
Vibrio Fast Detection Technique is to meet to Aquatic product disease control and Safety of Aquatic Products, particularly early diagnosiss technology and means.Mesh
Before, carry out the research of a large amount of vibriosises immunoassay technologies both at home and abroad, but be to prepare many grams using full vaccine mostly
, there is lacking of the aspects such as cross reaction, specificity and susceptiveness in grand antibody or polyclonal antibody and the method for quick set up
Fall into.
Over nearly 10 years, China carries out, compared with in-depth study, also building to the Fast Detection Technique of some major diseases and method
Some method for quickly detecting for some pathogen are found.The detection method set up mainly has immunology, pcr and nucleic probe
Deng technical method.Constantly it is applied to aquatic health cultivation, the immunology detection technical side of pathogen with immunology principle
The foundation of method is subject to the extensive concern of researchers.In recent years, some pathogenic bacterium vibrio Fast Detection Technique methods are also set up, main
Including immune labeled methods such as indirect elisa, sandwich elisa, dot-elisa, fluorescent antibodys.However, above-mentioned detection technique
Means are required for certain instrument and equipment, and operate more complicated, have significant limitation in popularization and application；Moreover, above
Research is mostly to be the method for quick set up for immune means with the polyclonal antibody of mammal acquisition or monoclonal antibody,
But mammal source (as rabbit, sheep etc.) antibody, easily there are multiple non-specific cross-reactions, cause false positive, be subject to
Certain restriction.
Content of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of gold colloidal-yolk antibody of vibrio parahaemolytious is fast
Fast Test paper and preparation method.
To achieve these goals, the technical solution used in the present invention is: a kind of yolk antibody of anti-vibrio parahaemolytious
Preparation method is it is characterised in that comprise the following steps: 1. immunogenic preparation, and immunogenic preparation is included outside vibrio parahaemolytious
Memebrane protein (ompk) and the preparation of hemolysin (tdh) antigen；2. above-mentioned vibrio parahaemolyticus outer membrane protein and hemolysin antigen are divided
It is not inoculated on healthy laying hen；3. laying hen, after immunity, is collected egg, and extracts pure yolk liquid, pure yolk liquid is carried out successively
Water extraction, saturated ammonium sulphate method and sephadexg-75 sephadex chromatography extract and respectively obtain pure secondary haemolysis arc
Bacterial outer membrane protein (ompk) yolk antibody and hemolysin (tdh) yolk antibody.
Further, the preparation of described vibrio parahaemolyticus outer membrane protein includes being set according to vibrio parahaemolytious ompk conserved sequence
Meter primer, the gene order of forward primer and downstream primer be respectively as follows: ompk-f (5 '-atggattactctgacggcg-3 ') and
Ompk-r (5 '-ttagaacttgtaagttactgc-3 '), pcr expand vibrio parahaemolyticus outer membrane protein ompk gene, by base
Because of recombination to construct recombiant plasmid pet28a-ompk, recombiant plasmid pet28a-ompk induction is proceeded to competent escherichia coli cell
Inducing culture, by purified for the supernatant protein of expression of recombinant plasmid, concentrate after, mix with adjuvant according to a certain percentage, be homogenized, make
Standby ompk antigen.
Further, vibrio parahaemolytious tdh gene order (m10069) design primer, forward primer and downstream primer are respectively
For: tdh-f (5 '-atgaaacaccaatattttgc-3 ') and tdh-r (5 '-ttattgttgatgtttacatt-3 '), pcr expand
Increase vibrio parahaemolytious tdh gene, using gene recombinaton construction recombination plasmid pet28a-tdh, recombiant plasmid pet28a-tdh is lured
Lead and proceed to competent escherichia coli cell inducing culture, the supernatant protein of just expression of recombinant plasmid is purified, concentrate after, according to
Certain proportion is mixed with adjuvant, is homogenized, and makes tdh antigen.
Further, the described purification process to pure yolk liquid, comprises the following steps: adds 9 times of bodies in pure yolk liquid
Long-pending distilled water, adjusts 10000r/min centrifugation 20min after 5.2,4 DEG C of standing 12h of ph, collect supernatant to obtain crude extract is chicken
Water soluble ingredient in yolk, then Deca equal-volume saturated ammonium sulfate solution make its saturation reach 50%, 4 DEG C stand overnight after from
Gains in depth of comprehension, to precipitate, make its saturation reach 33% with Deca saturated ammonium sulfate solution after equal-volume distillation water dissolution, 4 DEG C of standing 1h
Centrifugation is precipitated thing afterwards, is dissolved with former 1/2 volume phosphate buffered saline(PBS), and uses bag filter desalination, then through gel chromatography column
It is further purified, lyophilization, -20 DEG C of preservations.
Another technical scheme of the present invention is: a kind of preparation method of gold colloidal-yolk antibody complex, and its feature exists
In comprising the following steps: tdh yolk antibody is prepared using described claim 1-4 preparation method, then, by citric acid three
Sodium reduction prepares gold colloidal, adds k in colloid aurosol2co3Adjust ph, while stirring, be slowly added dropwise yolk antibody molten
Liquid, after stirring standing, adds bsa solution to prevent coagulation, then by ultracentrifugation, solution is carried out purification, obtain the glue concentrating
Body gold-yolk antibody complex.
Further, take 20ml colloid aurosol, add 1%k2co3240 μ l adjust ph, while stirring, are slowly added dropwise
Yolk antibody solution, stirs 10-30min.After standing 30min, add 10%bsa to final concentration of 1%, stir 10-20min.
Then solution is carried out purification by ultracentrifugation, 10000r/min is centrifuged 60min, abandons supernatant, precipitation gold labeling antibody buffers
Liquid dissolves, and repeats secondary centrifuging, finally resuspended with former 1/20 volume gold labeling antibody buffer, and the gold mark yolk obtaining concentrating resists
Body.
Further, the preparation method of described gold colloidal, comprises the following steps: takes clean conical flask, adds 0.01%
Gold chloride 100ml, heated and boiled, it is rapidly added 1% trisodium citrate aqueous solution 1.5ml, continues to boil, observe solution colour and become
Change, when being changed into claret completely, take out, be cooled to room temperature, ultra-pure water complements to 100ml.
Another technical scheme of the present invention is: a kind of gold colloidal of vibrio parahaemolytious-yolk antibody quick detection test paper,
It is characterized in that: include nitrocellulose filter (nc film), colloidal gold pad, viscosity pvc base plate, sample pad, absorbent filter, detection line
T () and nature controlling line (c), described colloidal gold pad is equipped with gold mark vibrio parahaemolytious tdh yolk antibody, described detection line and Quality Control
Line is obtained respectively by vibrio parahaemolytious ompk yolk antibody and rabbit anti-chicken igg, described vibrio parahaemolytious ompk yolk antibody and pair
Preparation method as described in claim 1-4 any one claim for the hemolysis vibrion tdh yolk antibody is obtained, and gold mark yolk resists
Preparation method described in described claim 6 for the body is obtained.
Further, described test strips include nc film, colloidal gold pad, viscosity pvc base plate, sample pad, absorbent filter, described
Nc film sticks at the position of 21-46mm on viscosity pvc base plate, detection line upper, nature controlling line under；Colloidal gold pad sticks at viscosity pvc
The position of 12mm on base plate, upper end is pressed on the lower end of nc film, overlapping 2mm, and sample pad sticks on viscosity pvc base plate, and upper end is pressed on
The lower end of colloidal gold pad, overlapping 3mm, absorbent filter sticks on viscosity pvc base plate, and lower end is pressed on the upper end of nc film, overlapping 2mm.
Another technical scheme of the present invention is: a kind of system of the vibrio parahaemolytious quick detection test paper based on yolk antibody
Preparation Method it is characterised in that: using described in described claim 1-4 preparation method be obtained ompk yolk antibody and tdh
Yolk antibody, the gold mark tdh yolk antibody being obtained by the preparation method described in claim 5, first, by doubling dilution gold mark
Antibody, and be taped against in colloidal gold pad, the gold colloidal having sprayed band is placed in 37 DEG C of dryings；Secondly, with buffer solution by pair
Hemolysis vibrion ompk yolk antibody (t) and anti-chicken igg (c) of rabbit are diluted to suitable concn, respectively as detection line and nature controlling line, will
Nc film, colloidal gold pad, viscosity pvc base plate, sample pad, absorbent filter are overlapped the to each other by viscosity end liner and stick into one, become
Complete test strips.
Using such scheme, the present invention, with vibrio parahaemolytious as research tool, is immunity using outer membrane protein and hemolysin
The former Rapid detection test strip prepared yolk antibody, develop vibrio parahaemolytious, and carry out Applied D emonstration in aquaculture area.This will
For carrying out early stage quick detection and the prevention and control of marine cultured animal kinds of pathogenic vibrio, and the control of marine product Safety of Food Quality carries
For technical support；The preventing and treating of the disease causing for marine cultured animal vibrio parahaemolytious lays the foundation.
The invention will be further described below in conjunction with the accompanying drawings.
Brief description
Fig. 1 is vibrio parahaemolytious schematic diagram in tcbs culture medium:
Fig. 2 is vibrio parahaemolytious ompk amplification:
The expression of Fig. 3 restructuring ompk, purification and westem blotting detection；
Fig. 4 vibrio parahaemolytious tdh gene amplification result；
Qualification result after Fig. 5 pet28a-tdh enzyme action；
Fig. 6 restructuring tdh expression and purification and detection；
The sds-page electrophoresis pattern of the igy that Fig. 7 distinct methods extract；
Tetra- kinds of methods of Fig. 8 extract the od value (initial dilution is 1: 10) of igy；
Fig. 9 ammonium sulfate method sds-page electrophoretogram after purification；
The gel elution collection of illustrative plates of Figure 10 igy；
Figure 11 difference purification process igy gel electrophoresis figure；
The potency ratio of Figure 12 difference yolk antibody is relatively；
The colloidal gold solution of Figure 13 trisodium citrate reduction method preparation；
Figure 14 albumen is combined optimum k with gold colloidal2co3The mensure of consumption；
Figure 15 is combined the mensure of the most suitable antibody protein binding capacity with gold colloidal；
The gold labeling antibody that Figure 16 prepares；
The susceptiveness based on the vibrio parahaemolytious colloidal gold strip of yolk antibody for the Figure 17；
The specificity based on the vibrio parahaemolytious colloidal gold strip of yolk antibody for the Figure 18.
Specific embodiment
It is described with reference to the drawings and content of the invention, the specific embodiment of the present invention is as follows, including following components:
(1) immunogenic preparation
1 materials and methods
1.1 material
1.1.1 bacterial strain
As shown in figure 1, strains tested is vibrio parahaemolytious, separated preservation.Plasmid, competent escherichia coli cell are purchased from
Shanghai biological engineering company limited.
1.1.2 solution is prepared
1m hcl solution: graduated cylinder measures 8.36ml concentrated hydrochloric acid (or directly weighing 9.86g in the balance), pours into already equipped with about
In the beaker of 50-70ml pure water, it is stirred continuously, be settled to 100ml after cooling, after aseptic filtration, room temperature preservation.
Pbs buffer (ph 7.4,0.01m): weigh nacl 8.0g, kcl 0.2g, na2hpo41.42g,
kh2po40.27g, is dissolved in 600ml pure water, is settled to 1l, 121 DEG C of sterilizing 20min, room temperature preservation.
30%acrylamide: weigh acrylamide 290g, bis 10g, add 600ml pure water, fixed after stirring and dissolving
Hold to 1l, filter off impurity with 0.45 μm of filter membrane, 4 DEG C of preservations in brown bottle.
1.5mol/l tris-hcl buffer (ph 8.8): weigh 181.7g tris and be placed in 1l beaker, plus 800ml is pure
Water, is sufficiently stirred for dissolving, and is adjusted with concentrated hydrochloric acid and is settled to 1l after ph to 8.8.After autoclave sterilization, room temperature preservation.
1mol/l tris-hcl buffer (ph 6.8): 121.1g tris is dissolved in 900ml pure water, further according to required
Ph (at 25 DEG C) plus certain concentrated hydrochloric acid (11.6mol/l), with distilled water adjustment final volume to 1l, autoclave sterilization is after room temperature
Storage.
10% (w/v) ap solution: weigh 1g ap, add stirring and dissolving after 10ml pure water, preserve in 4 DEG C.
10%sds solution: weigh 10g high-purity sds and be dissolved in 80ml pure water, addend drips dense hcl adjusts constant volume after ph to 7.2
To 100ml, room temperature preservation.
5 × tris-gly buffer (sds-page electrophoretic buffer): weigh tris 15.1g, glycine 94g, sds
5.0g, adds 800ml pure water, stirring and dissolving, after be settled to 1l, room temperature preservation, 5 times of dilutions during use.
5 × sds-page loading buffer reagent: 1m tris-hcl (ph6.8) 1.25ml, sds 0.5g, bpb
25mg, glycerol 2.5ml, plus pure water is settled to 5ml.
Coomassie brilliant blue r-250 dyeing liquor: weigh 1g Coomassie brilliant blue r-250, add 250ml isopropanol to stir；
Add 100ml glacial acetic acid, stir；Add 650ml pure water, stir；After filter paper filtering, room temperature preservation.
Coomassie brilliant blue destaining solution: measure acetic acid 100ml, acetic acid 50ml, pure water 850ml, be sufficiently mixed rear room temperature preservation.
1.1.3 the making of culture medium
Zobell 2216e culture medium: peptone 5g, yeast leaches cream 1g, iron phosphate 0.01g, Chen Haishui 1l, adjusts ph extremely
7.6-7.8 (during configuration solid medium, adds 16g agar powder), 121 DEG C of sterilizing 20min after subpackage.
Tcbs culture medium: tcbs 83g, pure water 1l, mixed and boiled 3 times, directly poured into sterile petri dish, cooling is standby
With.
Nutrient broth: take peptone 10.0g, beef powder 3.0g, sodium chloride 5.0g in 1000ml pure water, agitating heating is boiled
Boil to being completely dissolved, adjust ph to 7.2 ± 0.2 (during configuration solid medium, adding 16g agar powder), 121 DEG C of high pressure after subpackage
Sterilizing 15min.
The preparation of 1.2 vibrio parahaemolytious inactivated vaccines
Vibrio parahaemolytious inoculation in zobell 2216e inclined-plane, after 28 DEG C of culture 24h, with 0.65% physiology salt that sterilizes
The lower lawn of washing, after adding final concentration of 0.3% formalin-inactivated 24h, detects inactivating efficacy with tcbs culture medium.Confirm thoroughly
After inactivation, with sterilizing, 0.65% physiology salt is washed 3 times, is made into final concentration of 1 × 108Cfu/ml vibrio parahaemolytious inactivated vaccine.
1.3 vibrio parahaemolytious omp and the preparation of tdh antigen
1.3.1 the preparation of specificity ompk vaccine
Primer is designed according to vibrio parahaemolytious ompk conserved sequence, forward primer and downstream primer are respectively as follows: ompk-f
(5 '-atggattactctgacggcg-3 ') and ompk-r (5 '-ttagaacttgtaagttactgc-3 '), pcr amplification is secondary molten
Blood vibrio outer membrane protein ompk gene.
After sequence verification, using gene recombination technology, the construction recombination plasmid (prokaryotic expression carrier of expression ompk gene
Pet28a-ompk), itpg induction proceeds to E. coli competent.Empirical tests, recombiant plasmid pet28a-ompk is in escherichia coli
Expressed.
Recombinant expressed supernatant protein, after histrap purification, concentration, measures ompk albumen using bradford method dense
Degree.And mix with adjuvant according to a certain percentage, be homogenized, prepare ompk subunit vaccine.
1.3.2 the preparation of specificity vibrio parahaemolytious tdh vaccine
Vibrio parahaemolytious tdh gene order (m10069) according to report design primer, forward primer and downstream primer
It is respectively as follows: tdh-f (5 '-atgaaacaccaatattttgc-3 ') and tdh-r (5 '-ttattgttgatgtttacatt-3 '),
Pcr expands vibrio parahaemolytious tdh gene.
Compare through sequencing and balst, the sequence similarity announced with genbank is 99%.Using gene recombination technology, structure
Build recombiant plasmid (the prokaryotic expression carrier pet28a-tdh of expression tdh gene), itpg induction proceeds to escherichia coli top10 impression
State cell.Empirical tests, recombiant plasmid pet28a-tdh is expressed in escherichia coli.
2 results and analysis
The preparation of 2.1 vibrio parahaemolytious ompk
Application forward primer v-ompk-f (5 '-atggattactctgacggcg-3 ') and downstream primer v-ompk-r (5 '-
Ttagaacttgtaagttactgc-3 ') Successful amplification is to vibrio parahaemolyticus outer membrane protein ompk gene, about 750bp (Fig. 2).
Compare through sequencing and balst, the sequence similarity announced with genbank is 99%.
As Fig. 3, the prokaryotic expression carrier of ompk gene (is expressed using gene recombination technology success construction recombination plasmid
pet28a-ompk).Recombinant expressed supernatant protein, after histrap purification, concentration, measures ompk using bradford method
Protein concentration.And mix with adjuvant according to a certain percentage, be homogenized, make ompk subunit vaccine.
The preparation of 2.2 vibrio parahaemolytious tdh
Application forward primer tdh-f (5 '-atgaaacaccaatattttgc-3 ') and downstream primer tdh-r (5 '-
Ttattgttgatgtttacatt-3 ') Successful amplification is to vibrio parahaemolytious tdh gene, pcr product, about 570bp (Fig. 4).Warp
Sequencing and balst compare, and the sequence similarity announced with genbank is 99.6%.Weight is successfully built using gene recombination technology
Group plasmid (the prokaryotic expression carrier pet28a-tdh of expression tdh gene), visible two bands after recombiant plasmid double digestion, one is
Carrier, a size is about the purpose band (Fig. 5) of 570bp.
Recombiant plasmid pet28a-tdh is successfully proceeded to competent escherichia coli cell, after inducing culture, exists after testing
Protein band occurs at 27kd and is consistent (Fig. 6) with purpose band.Culture fluid supernatant protein enters to pass through histrap after purification, carries out
Western blotting detects, shows that 27kd albumen occurs specific reaction (Fig. 6).Recombiant protein and according to a certain percentage with
Adjuvant mixing, homogenate, make tdh subunit vaccine.
(2) refine the preparation of anti-vibrio parahaemolytious yolk antibody
1 materials and methods
1.1 experiment material
Test bacterial strain: vibrio parahaemolytious (vibrio parahaemolyticus) separate for this experiment and identify；Experiment
It is spf sea orchid chicken with laying hen.
The reagent such as Freund's complete adjuvant, incomplete Freund's adjuvant, peg-6000 are purchased from Shanghai and give birth to the limited public affairs of work biological engineering
Department, sephadex g-75 gel is purchased from sigma company.
The preparation of 1.2 antigens
Antigen is the vibrio parahaemolytious inactivated vaccine of above-mentioned preparation, ompk and tdh.
The immunity of 1.3 laying hens
Take standby above-mentioned antigen, 0.1mg recombiant protein ompk and tdh adds 1ml Freund's complete adjuvant or Freund not complete
Full adjuvant, vibrio parahaemolytious suspension 1 × 108Cfu/ml adds isopyknic Freund's complete adjuvant or incomplete Freund's adjuvant.Point
After not fully emulsified, immune health sea orchid laying hen.The lower 4 points of subcutaneous injections of breast, neck, the wing are taken in immunity, and first immunisation injection is containing not
The antigen of family name's Freund's complete adjuvant, dosage is every 1.0ml.1st booster immunization, every 1.5ml after 10d；Carry out the 2nd time after 20d
Booster immunization, every 2.0ml；Start within 1 week after 2nd booster immunization to collect egg, determined whether into traveling according to concrete potency
The 3rd booster immunization of row.The antigen containing incomplete Freund's adjuvant for injection during booster immunization.Set non-immunized controls group simultaneously.
The separation of 1.4 yolk antibody water-soluble components (wsf)
Two exempt from after collect egg, with clear water clean egg surface contaminants, dry, numbering latter 4 DEG C save backup.Water is taken to dilute
Method carries out crude separation to Yolk immunoglobulin igy: carefully knocks eggshell open, exhausts Ovum Gallus domesticus album with syringe, adds sterile purified water
Clean Ovum Gallus domesticus album, after egg yolk is rolled on filter paper, syringe punctures vitellinae membrana, collects yolk liquid.Plus 9 times of long-pending going out of yolk liquid
Bacterium distilled water, adjusts ph to 5.2-5.3, stirs latter 4 DEG C and stand overnight, 4 DEG C, and 10000r/min is centrifuged 25min, takes the supernatant to be
For wsf.
The secondary separation of 1.5 anti-vibrio harveyi specific igy
This experiment compares phynol method, cold Ethanol Method, ammonium sulfate method and 4 kinds of methods of caprylic acid-ammonium and resists Kazakhstan arc
Bacterium yolk antibody carries out the effect of secondary purification.Respectively from taking yolk liquid 2ml with 1 piece of egg, carry out carrying of 4 kinds of distinct methods
Take, if 3 repetitions.Concrete grammar is as follows:
Phynol method: stirred at 4: 1 by volume with the normal saline containing 3% phenol and yolk, stand 10min.4 DEG C,
4000r/min, is centrifuged 10min, removes precipitation, then is mixed with supernatant with equivalent 50% cold ethanol precipitation, 4000r/min, from
Heart 15min, takes resolution of precipitate in the pbs of original volume.
Ammonium sulfate method: yolk liquid is diluted with distilled water 1: 9, adjusts 10000r/min centrifugation after 5.2,4 DEG C of standing 12h of ph
20min, collect supernatant to obtain crude extract is wsf.Then Deca equal-volume saturated ammonium sulfate solution makes its saturation reach 50%,
4 DEG C stand overnight after centrifugation be precipitated thing, with equal-volume distillation water dissolution after Deca saturated ammonium sulfate solution make its saturation
Reach 33%, centrifugation after 4 DEG C of standing 1h is precipitated thing, with former 1/2 volume pbs buffer solution, and use bag filter desalination.
Cold Ethanol Method: 95% ethanol is placed in after -20 DEG C of pre-cooling 2h, slowly adds with 1: 1 ethanol of ethanol volume ratio by wsf
Enter in wsf, 4 DEG C of stirring 30min, after precipitation is complete, 4 DEG C, 22000r/min is centrifuged 20min, abandons supernatant.Precipitate is dissolved in
So as to become homogeneous suspension in 25mmol/lnacl solution.4 DEG C of filtrations, remove lipoprotein pellet therein, collect filter
Liquid.Then add 50% cold ethanol make its final concentration of 30%, 4 DEG C, 22000r/min, be centrifuged 20min, precipitation distillation
Water dissolution.
Caprylic acid-ammonium: to being slowly added to the caprylic acid that whole degree is 1% in wsf, stir 10min, put in refrigerator
Freezing 10min, sucking filtration obtains igy solution.Then Deca equal-volume saturated ammonium sulfate solution makes its saturation reach 50%, 4 DEG C of standings
After overnight, 4 DEG C, 10000r/min, centrifugation 20min abandons supernatant, is made with Deca saturated ammonium sulfate solution after distilled water dissolution precipitation
Its saturation reaches 33%, 4 DEG C after 4 DEG C of standing 1h, 10000r/min, and supernatant is abandoned in centrifugation 20min centrifugation, is delayed with former 1/2 volume pbs
Rush liquid dissolving, and use bag filter desalination.
1.4 igy gel chromatographies
With the yolk antibody solution of saturated ammonium sulfate method preparation, after bag filter desalination.Sephadex g-75 gel chromatography
Post is further purified, lyophilization, -20 DEG C of preservations, and passes through sds-page electrophoresis detection Sample Purification on Single effect.
The indirect elisa of 1.5 yolk antibody igy potency measures
Elisa operational approach is indirectly:
(1) be coated buffer dilution specific antigen be vibrio parahaemolytious, final concentration 108Cfu/ml, coated elisa plate,
100 μ l, 4 DEG C overnight；
(2) plate 3 times washed by cleaning mixture, and confining liquid is closed, 100 μ l/ holes, 37 DEG C of incubation 1h；
(3) plate 3 times washed by cleaning mixture, every hole add by the secondary separation than dilution after specificity igy (initial concentration is
0.97mg/ml).It is simultaneously introduced negative control (non-specific igy) and blank, 100 μ l/ holes, 37 DEG C of incubation 2h；
(4) plate 3 times washed by cleaning mixture, plus peroxidase hrp labelling two resists, 100 μ l/ holes, 37 DEG C of incubation 1h；
(5) cleaning mixture washes plate 3 times, plus tmb working solution (matching while using), 100 μ l/ holes, and shady place reacts 20min；
(6) add terminate liquid (2m h2so4), 50 μ l/ holes, microplate reader detection od value (wavelength 450nm)；
(7) analytical data, dilution factor corresponding to 2.1 times of negative control value for the od value is its potency.
2 results and analysis
The igy sds-page electrophoresis result that 2.1 distinct methods extract
By sds-page electrophoresis pattern (Fig. 7), the heavy chain molecule amount of igy is 67kd, and light chain molecule amount is 23kd.
The protein band of yolk wsf at most, is used alone sad method and takes second place, and phynol method and cold Ethanol Method occupy middle, ammonium sulfate method and
Protein band contained by caprylic acid-ammonium is the most clear, and foreign protein is less.M albumen mark in Fig. 7；1 yolk wsf；2 phenol
Method；3 cold Ethanol Method；4 sad methods；5 ammonium sulfate methods；6 caprylic acid-ammoniums.
The od value that 2.2 distinct methods extract igy compares
Surveyed with indirect elisa method and compare the potency that 4 kinds of methods extract igy.As seen from Figure 8, phynol method extracts
Potency is minimum, is secondly cold Ethanol Method, and it is almost same with the igy potency of simple ammonium sulfate extraction that caprylic acid-ammonium extracts igy
Step.
2.3 ammonium sulfate methods slightly purify character and the total protein content of igy
It is odorlessness residual with the igy that ammonium sulfate method is extracted, after measured, protein concentration is 0.911 ± 0.09mg/ml.Logical
Cross sds-page electrophoresis pattern (Fig. 9) to understand, the heavy chain molecule amount of igy is 67kd, and light chain molecule amount is 23kd.Yolk wsf's
Protein band is a lot, and also ratio is more visible for the band of ammonium sulfate method purification, and foreign protein reduces a lot.M albumen mark in Fig. 9；1 yolk
wsf；2 ammonium sulfate methods slightly carry yolk antibody.
2.4 gel chromatography igy
Sample after peg-6000 after secondary for ammonium sulfate saltouing is concentrated passes through sephaedex g-75 gel column, automatically receives
Collection, draws elution curve.Result is shown in Figure 10.
2.5 sds-page detection igy purity
After solution after the aqueous composition of anti-vibrio parahaemolytious specificity igy, first time ammonium sulfate, second ammonium sulfate
After solution, gel chromatography, solution is sample, purifies situation with sds-page electrophoresis detection igy, and result is shown in Figure 11.In Figure 11, m
mark；1wsf；After 2 first time ammonium sulfate；After 3 second ammonium sulfate；After 4 gel chromatographies.
After 2.6 different purification process, igy antibody titer compares
The igy antibody titer obtaining after the water extraction of same protein content, ammonium sulfate precipitation method and gel chromatography, gel layer
After analysis, the igy potency of purification preferably, is best suitable for for research and development detection product.
(3) specificity of the yolk antibody of different immunogen preparations compares
In yolk antibody preparation process, with Indirect Elisa monitoring different antibodies titre change, investigate different antibodies neutralization
Ability.Anti- vibrio parahaemolytious fkc yolk antibody, anti-vibrio parahaemolytious tdh yolk antibody and anti-vibrio parahaemolytious ompk yolk resist
Body reaches the optimal potency time, optimum antibody potency is held time, and antibody titer has differences (Figure 12).
Isolating and purifying of yolk antibody is carried out using methods such as water extraction, ammonium sulfate precipitation, gel chromatographies, obtains high-purity
Anti- vibrio parahaemolytious fkc yolk antibody, anti-vibrio parahaemolytious tdh yolk antibody and anti-vibrio parahaemolytious ompk yolk antibody,
Sds-page have detected and isolates and purifies effect, purity difference with insignificance.Save backup after the yolk antibody lyophilization of preparation.
Simultaneously harmful with other common aquatic pathogenic bacteria and food using neutralizing the different yolk antibodies of experimental analysiss
The cross reaction situation of antibacterial, finds that anti-vibrio parahaemolytious fkc yolk antibody has certain intersecting with vibrio harveyi, Vibrio campbellii
Reaction, there is cross reaction in anti-vibrio parahaemolytious ompk yolk antibody and vibrio harveyi, anti-vibrio parahaemolytious tdh yolk antibody is no
Cross reaction.
According to above-mentioned comparative result, anti-vibrio parahaemolytious tdh yolk antibody and anti-vibrio parahaemolytious ompk yolk is selected to resist
Body is as capture antibody and the detection antibody prepared needed for colloidal gold fast detecting test paper strip.
(4) preparation of gold mark yolk antibody and reaction condition screening
1 materials and methods
1.1 experiment material
Gold chloride, trisodium citrate, bsa are purchased from sigma company；Other conventional reagent are purchased from Shanghai and give birth to work biological engineering
Company limited.
The preparation of 1.2 yolk antibodies
The vibrio parahaemolytious tdh yolk antibody of above-mentioned preparation.
The cleaning of 1.3 glass containers
The pollution of glass surface can disturb the generation of gold grain, and all glass containers are using front through overpickling silication.Method
It is that the first glass container using is soaked in 1 minute in the chloroformic solution of 5% dichlorodimethylsilane, distilled water after drying at room temperature
Rinse, re-dry is standby.The vessel of Reusability, with DDW drip washing after gold solution rinse, replace silicidation.
1.4 prepare gold colloidal using trisodium citrate reduction method
Take the clean conical flask of a 250ml, add 0.01% gold chloride 100ml, heated and boiled.Rapid as needed
Add 1% trisodium citrate aqueous solution 1.5ml, continue to boil, observe solution colour change, when being changed into claret completely, take
Go out, be cooled to room temperature, ultra-pure water complements to 100ml.
1.5 albumen are combined optimum k with gold colloidal2co3The mensure of consumption
Take 8 1.5ml ep pipes, each addition 1ml gold colloidal, and press table 2-1 and add 1%k2co3, after stirring, add
Antibody 10 μ g, adds 5% nacl solution 50 μ l after stirring evenly 20min, place 1h, the change of observing colloid gold color under room temperature.
Table 1 optimum k2co3Consumption determines
1.6 optimum antibody protein binding capacities
Take 6 1.5ml ep pipes, each addition 1ml gold colloidal, and be separately added into 1%k2co312 μ l adjust ph, stir
Afterwards, add antibody by table 2, after stirring evenly 20min, add 5% nacl solution 50 μ l, under room temperature, place 1h, observing colloid gold color
Change.
Table 2 optimum antibody protein binding capacity determines
1.7 gold colloidals-antibody complex stabilizer
For preventing the coagulation of gold labeling antibody, the antibody after 1ml labelling is taken to be slowly added to 10%bsa100 μ l.
The preparation of 1.8 gold colloidals-antibody complex and purification
Take 20ml colloid aurosol, add 1%k2co3240 μ l adjust ph, while stirring, are slowly added dropwise yolk antibody molten
Liquid, stirs 10-30min.After standing 30min, add 10%bsa to final concentration of 1%, stir 10-20min.Then by solution
Purification is carried out by ultracentrifugation, 10000r/min is centrifuged 60min, abandon supernatant, precipitation uses gold labeling antibody buffer solution, repeats
Secondary centrifuging, finally resuspended with former 1/20 volume gold labeling antibody buffer, obtain the gold mark yolk antibody concentrating.
2 results
The preparation of 2.1 gold colloidals
Aqueous solution of chloraurate is faint yellow, after heating, reacts with trisodium citrate aqueous solution, solution is rapidly from yellowish
Color is changed into black, is then changed into lavender, eventually becomes stable claret as shown in figure 13.
2.2 optimum k2co3Mensure
With k2co3After adjusting the ph of each colloidal gold solution, add the antibody of same volume, after mixing 20min, add nacl molten
Liquid.Standing is observed, except k2co3Addition is that 0-9 μ l group colloidal gold solution color is changed into blue from claret, remaining each pipe solution
All do not change, see Figure 14.Result shows, every 1ml colloidal gold solution adds k2co3During solution 12 μ l, gold colloidal-antibody complex
Relatively stable.In Figure 14,1-8 is respectively k2co3Volume 0 μ l, 1 μ l, 3 μ l, 6 μ l, 9 μ l, 12 μ l, 15 μ l and 18 μ l.
The mensure of 2.3 the most suitable antibody protein binding capacities
As shown in figure 15, when antibody protein content 5 μ g or during more than 5 μ g in 1ml colloidal gold solution, colloidal gold solution face
Color does not change, and forms stable gold colloidal-antibody complex, in Figure 15 1-6 be respectively antibodies amount 10 μ g, 5 μ g,
2.5 μ g, 2 μ g, 1 μ g and 0 μ g.
The preparation of 2.4 gold labeling antibodies
Prepare gold labeling antibody according to optimal condition to obtain stablizing bright deep claret solution, see Figure 16.
(5) the vibrio parahaemolytious Rapid detection test strip based on yolk antibody is developed and performance detection
1 materials and methods
1.1 experiment material
Test bacterial strain: vibrio parahaemolytious inactivated vaccine (108cfu/ml).
The vibrio parahaemolytious tdh gold labeling antibody of above-mentioned preparation, vibrio parahaemolytious ompk yolk antibody and rabbit anti-chicken igg resist
Body.
Nc film, colloidal gold pad, viscosity pvc base plate, sample pad, absorbent filter etc..
1.2 paving gold
Doubling dilution gold labeling antibody, is taped against in colloidal gold pad, and the gold colloidal having sprayed band is placed in 37 DEG C of dryings.
1.3 antigen/antibody line
With buffer solution, vibrio parahaemolytious ompk yolk antibody (t) and anti-chicken igg (c) of rabbit are diluted to suitable concn, point
Not as detection line and nature controlling line.
1.4 test strips assemblings
By the various solid phase materials after processing, overlapped the to each other by viscosity end liner and stick into one, become complete reagent paper
Bar, detailed process is as follows:
(1) nc film is sticked at the position of 21-46mm on viscosity pvc base plate, c line upper, t line under；
(2) colloidal gold pad is sticked at the position of 12mm on viscosity pvc base plate, upper end is pressed on the lower end of nc film, overlapping 2mm；
(3) sample pad is sticked on viscosity pvc base plate, upper end is pressed on the lower end of colloidal gold pad, overlapping 3mm；
(4) absorbent filter sticks on viscosity pvc base plate, and lower end is pressed on the upper end of nc film, overlapping 2mm；
(5) band that assembling shapes cuts into the wide test strips of 4mm, and is stored in dry environment.
1.5 colloidal gold immuno-chromatography test paper strip testing result judges
With 108As positive control, pbs buffer is negative control to cfu/ml vibrio parahaemolytious bacteria suspension.By test strips
In insertion contrast solution and sample suspension, after sample chromatography finishes, observe testing result.In test strip reaction zone, t carries and c
At band, one red tape of each appearance, then it represents that having vibrio parahaemolytious in measuring samples, is judged to the positive, if occurring one only at c band
Red tape, then be judged to feminine gender；If c band occurs without red tape then it represents that test strip lost efficacy.
The sensitivity of 1.6 colloidal gold immuno-chromatography test paper strip detections
Take vibrio parahaemolytious culture, after formalin-inactivated, be diluted to the bacteria suspension of variable concentrations: 109cfu/ml、108cfu/
ml、107cfu/ml、106cfu/ml、105cfu/ml、104cfu/ml、103cfu/ml、102cfu/ml、101Cfu/ml and
100Cfu/ml, observes testing result after sample chromatography finishes.
The specificity of 1.7 colloidal gold immuno-chromatography test paper strip detections
With vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, Vibrio splindidus, vibrio cholera, Vibrio aestuarianus, slow love
Moral China bacterium, Aeromonas hydrophila, aeromonas salmonicida, streptococcus agalactiae, escherichia coli, staphylococcus aureuses, Aerugo are false single
Born of the same parents bacterium, pseudomonas putida, pseudomonas fluorescens, Salmonella typhimurium, bacillus subtilises, bacillus thuringiensiss and wax
The culture of sample bacillus cereuss totally 20 plants of non-vibrio parahaemolytious bacterial strains carries out test strips specific detection.Adjust each bacteria suspension concentration
To 1 × 108Cfu/ml, observes testing result after sample chromatography finishes.
The stability of 1.8 ELISA test strips
4 DEG C of Refrigerator stores will be placed on after the test strips packaging of preparation, take out test strips within every 30 days and be measured, test altogether
To experiment the 180th day, observe the impact to detection paper for the different time.Under identical experiment condition with 0cfu/ml,
103Cfu/ml and 106The vibrio parahaemolytious of cfu/ml are tested.
2 results
The sensitive analysis of 2.1 test strips
As shown in figure 17, the sensitivity that colloidal gold strip detects to positive sample is 102cfu/ml.In Figure 17,1-9 divides
Wei not vibrio parahaemolytious 108cfu/ml、107cfu/ml、106cfu/ml、105cfu/ml、104cfu/ml、103cfu/ml、
102cfu/ml、101Cfu/ml and 100cfu/ml.
The specificity analyses of 2.2 test strips
By the test strips after assembling and vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, Vibrio splindidus, cholera arc
Bacterium, Vibrio aestuarianus, Wdwardsiella tarda, Aeromonas hydrophila, aeromonas salmonicida, streptococcus agalactiae, escherichia coli, golden yellow
Staphylococcuses, Pseudomonas aeruginosa, pseudomonas putida, pseudomonas fluorescens, Salmonella typhimurium, bacillus subtilises,
Bacillus thuringiensiss and bacillus cereuss reaction, all do not produce positive reaction, and result shows that test strips specificity is high, sees figure
18.In Figure 18,1-2 is vibrio parahaemolytious, and 3-22 respectively adds vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus, vibrio alginolyticus, brilliance
Vibrio, vibrio cholera, Vibrio aestuarianus, Wdwardsiella tarda, Aeromonas hydrophila, aeromonas salmonicida, streptococcus agalactiae, large intestine
Bacillus, staphylococcus aureuses, Pseudomonas aeruginosa, pseudomonas putida, pseudomonas fluorescens, Salmonella typhimurium, withered
Careless bacillus cereuss, bacillus thuringiensiss and bacillus cereuss.
After measured, in 180 days 4 DEG C of Refrigerator stores test strips to 0cfu/ml, 103Cfu/ml and 106The pair of cfu/ml is molten
Three kinds of samples of blood vibrio all can normal table develop the color, and performance no significant difference has good stability.Under the same conditions,
The result that different experiments personnel are tested simultaneously does not have difference, and the test result of same personnel's different time is also consistent,
There is good repeatability.
The present invention adopts yolk antibody as immune means, association colloid technology for gold, have developed the early stage of vibrio parahaemolytious
Rapid detection test strip, has the advantages that sensitive, convenient use, can be widely used in the quick inspection of aquatic animal early stage
Survey, have a good application prospect.Its Demonstration Application be beneficial to early discovery hide cause of disease, reduce to greatest extent because of disease
The direct economic loss causing, is conducive to the increasing both production and income of raiser；Be conducive to improving aquatic product quality it is ensured that food matter simultaneously
Amount safety, has notable social benefit and ecological environment benefit.The present invention is not limited to above-mentioned specific embodiment, this area one
As technical staff according to present disclosure, the present invention can be implemented using other multiple specific embodiments, or
The design structure of every employing present invention and thinking, do simple change or change, both fall within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of the yolk antibody of anti-vibrio parahaemolytious is it is characterised in that comprise the following steps: 1. immunogenic
Preparation, immunogenic preparation includes vibrio parahaemolyticus outer membrane protein (ompk) and the preparation of hemolysin (tdh) antigen；2. will be above-mentioned
Vibrio parahaemolyticus outer membrane protein and hemolysin antigen are inoculated on healthy laying hen respectively；3. laying hen, after immunity, collects egg, and
Extract pure yolk liquid, pure yolk liquid is carried out successively with water extraction, saturated ammonium sulphate method and sephadexg-75 glucosan and coagulates
Glue-line analysis extraction respectively obtains pure vibrio parahaemolyticus outer membrane protein (ompk) yolk antibody and hemolysin (tdh) yolk resists
Body.
2. the yolk antibody of anti-vibrio parahaemolytious according to claim 1 preparation method it is characterised in that: described pair is molten
The preparation of blood vibrio outer membrane protein includes designing primer, forward primer and downstream primer according to vibrio parahaemolytious ompk conserved sequence
Gene order be respectively as follows: ompk-f (5 '-atggattactctgacggcg-3 ') and ompk-r (5 '-
Ttagaacttgtaagttactgc-3 '), pcr expands vibrio parahaemolyticus outer membrane protein ompk gene, is built by gene recombinaton
Recombiant plasmid pet28a-ompk, recombiant plasmid pet28a-ompk induction is proceeded to competent escherichia coli cell inducing culture,
By purified for the supernatant protein of expression of recombinant plasmid, concentrate after, mix with adjuvant according to a certain percentage, be homogenized, prepare pair haemolysis
Vibrio ompk antigen.
3. the yolk antibody of anti-vibrio parahaemolytious according to claim 1 preparation method it is characterised in that: described pair is molten
The preparation of blood vibrio tdh includes designing primer according to vibrio parahaemolytious tdh gene order (m10069), and forward primer and downstream are drawn
Thing is respectively as follows: tdh-f (5 '-atgaaacaccaatattttgc-3 ') and tdh-r (5 '-ttattgttgatgtttacatt-
3 '), pcr amplification vibrio parahaemolytious tdh gene, using gene recombinaton construction recombination plasmid pet28a-tdh, by recombiant plasmid
Pet28a-tdh induction proceeds to competent escherichia coli cell inducing culture, and just the supernatant protein of expression of recombinant plasmid is through pure
After change, concentration, mix with adjuvant according to a certain percentage, be homogenized, make vibrio parahaemolytious tdh antigen.
4. the yolk antibody of anti-vibrio parahaemolytious according to claim 1 preparation method it is characterised in that: described high-purity
The preparation process of degree yolk antibody comprises the following steps: the distilled water of 9 times of volumes of addition in pure yolk liquid, tune ph5.2,4 DEG C
10000r/min centrifugation 20min after standing 12h, collect supernatant to obtain crude extract is water soluble ingredient in Ovum Gallus domesticus Flavus, then drips
Plus equal-volume saturated ammonium sulfate solution makes its saturation reach 50%, 4 DEG C stand overnight after centrifugation be precipitated thing, steamed with equal-volume
After distilled water dissolving, Deca saturated ammonium sulfate solution makes its saturation reach 33%, and centrifugation after 4 DEG C of standing 1h is precipitated thing, with former 1/
2 volume phosphate buffered saline(PBS) dissolvings, and use bag filter desalination, after saltouing, peg-6000 concentrates, then through sephadexg-75 Portugal
Polysaccharide gel chromatographic column is further purified, lyophilization, -20 DEG C of preservations.
5. a kind of preparation method of gold colloidal-yolk antibody complex is it is characterised in that comprise the following steps: using described power
Profit requires 1-4 any one preparation method preparation tdh yolk antibody, then, prepares gold colloidal by trisodium citrate reduction method,
K is added in colloid aurosol2co3Adjust ph, while stirring, be slowly added dropwise yolk antibody solution, after stirring standing, add
Bsa solution prevents coagulation, then by ultracentrifugation, solution is carried out purification, and the gold colloidal-yolk antibody obtaining concentrating is combined
Thing.
6. gold colloidal according to claim 5-yolk antibody complex preparation method it is characterised in that: take 20ml glue
Body aurosol, adds 1%k2co3240 μ l adjust ph, while stirring, are slowly added dropwise yolk antibody solution, stir 10-
30min.After standing 30min, add 10%bsa to final concentration of 1%, stir 10-20min.Then by solution pass through hypervelocity from
The heart carries out purification, and 10000r/min is centrifuged 60min, abandons supernatant, precipitation gold labeling antibody buffer solution, repeats secondary centrifuging,
Finally resuspended with former 1/20 volume gold labeling antibody buffer, obtain the gold mark yolk antibody concentrating.
7. gold colloidal according to claim 5-yolk antibody complex preparation method it is characterised in that: described colloid
The preparation method of gold, comprises the following steps: takes clean conical flask, adds 0.01% gold chloride 100ml, heated and boiled, rapidly
Add 1% trisodium citrate aqueous solution 1.5ml, continue to boil, observe solution colour change, when being changed into claret completely, take
Go out, be cooled to room temperature, ultra-pure water complements to 100ml.
8. a kind of gold colloidal of vibrio parahaemolytious-yolk antibody quick detection test paper it is characterised in that: include nitrocellulose filter
(nc film), colloidal gold pad, viscosity pvc base plate, sample pad, absorbent filter, detection line (t) and nature controlling line (c), described colloidal gold pad
On be equipped with gold mark vibrio parahaemolytious tdh yolk antibody, described detection line and nature controlling line are by vibrio parahaemolytious ompk yolk antibody
And rabbit anti-chicken igg is obtained respectively, described vibrio parahaemolytious ompk yolk antibody and vibrio parahaemolytious tdh yolk antibody such as right will
The preparation method described in 1-4 any one claim is asked to be obtained, gold mark preparation described in described claim 6 for the yolk antibody
Method is obtained.
9. the gold colloidal of vibrio parahaemolytious according to claim 8-yolk antibody quick detection test paper it is characterised in that:
Described test strips include nc film, colloidal gold pad, viscosity pvc base plate, sample pad, absorbent filter, and described nc film sticks at viscosity pvc bottom
The position of 21-46mm on plate, detection line upper, nature controlling line under；Colloidal gold pad sticks at the position of 12mm on viscosity pvc base plate,
Upper end is pressed on the lower end of nc film, overlapping 2mm, and sample pad sticks on viscosity pvc base plate, and upper end is pressed on the lower end of colloidal gold pad, weight
Folded 3mm, absorbent filter sticks on viscosity pvc base plate, and lower end is pressed on the upper end of nc film, overlapping 2mm.
10. a kind of preparation method of the vibrio parahaemolytious quick detection test paper based on yolk antibody it is characterised in that: using by institute
State ompk yolk antibody and the tdh yolk antibody that the preparation method described in claim 1-4 any one is obtained, by claim
The gold mark tdh yolk antibody that preparation method described in 5 is obtained, first, by doubling dilution gold labeling antibody, and is taped against colloid
In gold pad, the gold colloidal having sprayed band is placed in 37 DEG C of dryings；Secondly, with buffer solution by vibrio parahaemolytious ompk yolk antibody
T anti-chicken igg (c) of () and rabbit is diluted to suitable concn, respectively as detection line and nature controlling line, by nc film, colloidal gold pad, viscosity
Pvc base plate, sample pad, absorbent filter are overlapped the to each other by viscosity end liner and stick into one, become complete test strips.
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| CN107202884A (en) * | 2017-06-16 | 2017-09-26 | 吉林大学 | Based on immunomagnetic beads and MnO2Nano-particle detects vibrio parahemolyticus kit |
| CN108992669A (en) * | 2018-08-27 | 2018-12-14 | 广州汇高生物科技有限公司 | Composite yolk antibody composition, aerosol inhalation solution agent and the preparation process and application for preventing and treating respiratory tract infection |
| CN110836975A (en) * | 2019-11-29 | 2020-02-25 | 扬州大学 | Colloidal gold plastic-sealed detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof |
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| CN107202884B (en) * | 2017-06-16 | 2019-03-19 | 吉林大学 | Based on immunomagnetic beads and MnO2Nanoparticle detects vibrio parahemolyticus kit |
| CN108992669A (en) * | 2018-08-27 | 2018-12-14 | 广州汇高生物科技有限公司 | Composite yolk antibody composition, aerosol inhalation solution agent and the preparation process and application for preventing and treating respiratory tract infection |
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| CN110836975B (en) * | 2019-11-29 | 2023-05-02 | 扬州大学 | Colloidal gold plastic package detection card for detecting vibrio parahaemolyticus TD toxin and preparation method and application thereof |
| CN114146213A (en) * | 2021-12-06 | 2022-03-08 | 广西博生生物科技有限公司 | Gel dressing of composite yolk globulin gamma protein and preparation method and application thereof |
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