CN109254156A - Detect immune-blotting method kit and its application of Pseudopleuronectes fish vitellogenin - Google Patents
Detect immune-blotting method kit and its application of Pseudopleuronectes fish vitellogenin Download PDFInfo
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Abstract
The invention discloses a kind of immune-blotting method kit for detecting Pseudopleuronectes fish vitellogenin and its applications.The kit contains Pseudopleuronectes fish-egg xanthan protein metabolism product lipovitellin sterling and its monoclonal antibody, when preparation, lipovitellin is purified into from Pseudopleuronectes fish-egg homogenate extracting solution first with gel filtration and ion exchange two-step chromatography, then pass through the monoclonal antibody of the immune obtained lipovitellin of hybridoma, and chromatographed using Hitrap Protein G, purifying obtains the anti-Pseudopleuronectes lipovitellin monoclonal antibody of mouse.Kit of the invention can be used for the screening of marine environment estrogen drugs, it being capable of vitellogenin in sensitive, convenient qualitative detection Pseudopleuronectes fish blood, epidermal mucus, liver organization and hepatocyte cultures liquid, lowest detection is limited to 0.75ng/mL, and the detection for China's immediate offshore area incretion interferent provides important evidence.
Description
Technical field
The invention belongs to ecological detection fields, and in particular to a kind of immunoblotting inspection for detecting Pseudopleuronectes fish vitellogenin
Test agent box and its application.
Background technique
((Persistent Organic Pollutants, POPs), it is difficult in the environment to refer to for persistence organic pollutant
To decompose, can long-term existence in the environment, the diffusion mobility of Global Scale can be carried out by various delivering paths, is passed through
Food chain accumulates amplification in vivo, and a kind of organic pollutant of toxic effect is generated to human body and environment.These pollutants
Be not that nature is inherently existing, but human industry's revolution bring product persistence organic pollutant to the mankind and
The harm of environment bring has become global problem.In order to solve this problem, United Nations Environment Programme (UNEP) and auspicious
Allusion quotation government holds plenipotentiary conference in the Stockholm co-anchor of Sweden on May 23rd, 2001, endorsed and is intended to prohibit
Only and/or limitation use 12 class persistence organic pollutants " Convention of Stockholm about persistence organic pollutant ".
The characteristic that POPs has following four important: (1) can in the environment enduringly exist；(2) it can be arrived by long-distance migration
Up to remote extremely low area.(3) harmful or toxic effects, POPs can be caused to the biology for contacting the substance under certain concentration
Mostly there is " three cause (carcinogenic, teratogenesis, mutagenesis) " effect；(4) it can be accumulated in food chain, the life to there is better nutritivity grade
Object impacts.Since POPs has the characteristics that low aqueous solubility, fat-solubility, POPs is caused to be enriched to biology from medium around
In vivo, and by the biological magnification of food chain reach poisoning concentration.
POPs is difficult to degrade in marine environment, distribution is wide, is easily enriched in vivo, very to the harmfulness of ecological environment
Greatly, POPs not only influences inhabiting and breeding for marine organisms, by the transmitting of food chain, also survives and lives to mankind itself
It threatens.Researches show that POPs concentration levels and not bery high for items, but since its bioconcentration can be passed by food chain
Enrichment is passed, so that bigger in the threat that the higher biology of food chain is subject to.In marine environment and other aquatic ecosystems,
The propagation chain of POPs is: the POPs in air be initially be absorbed by the micro-organisms → larger biology eat tiny organism → small fish it is edible compared with
Big biology → big fish swallowing little fish → is sometimes birds or the mankind eats big fish.The intracorporal POPs content of carnivore species will reach
As many as 10 times of POPs average content in its prey body.Which results in high POPs in most significant end food meat species body to contain
Amount.According to the report of Canadian (Environment Canada) tissue of environment, POPs pollutant reaches in the bird egg of edible fishes
25,000,000 times of POPs content in the water lived to fish itself.And be located at the mankind on biological chain top, then again these toxicity
It is exaggerated 70,000 times.
It is current to be badly in need of General Promotion ocean POPs real-time monitoring, pre-alerting ability, the pollution sources of sea-farming are studied,
The influence to aquatic product quality such as fish feed, mankind's activity and environmental factor is inquired into, is the quality monitoring of mariculture products
Management with marine environment provides scientific basis.Therefore it sets up quick, efficient, big flux and especially newly increases kind for POPs
Effective detection method of pollutant is to reinforce and improve the premise and base of detection and the risk control management to POPs in environment
Plinth.Studies have shown that most of POPs have environmental estrogens effect, it is environmental estrogens analog, therefore can be female by environment
Hormone biological detecting method is used for the biological detection of POPs.
The U.S., European Union and Japan set up the environmental estrogens screening and assessment system using fish as model organism in succession,
Middle vitellogenin (Vitellogenin, Vtg) has been used widely as important biological screening index.Fish ovum
Xanthan albumen is the precursor of livetin, is a kind of lipophosphoglycan albumen of macromolecule.Period of yolk formation, in the thorn of estrogen
Swash lower vitellogenin to be synthesized by liver, and through blood transportation into the ovary of development, cystovarian absorbs；In general, yolk is former
Albumen can only be arrived in the raun vivo detection of period of yolk formation, still, vitellogenin gene also be contained in milter and juvenile fish body,
Under the induction of environmental estrogens, vitellogenin can be also synthesized and secreted.Therefore, vitellogenin is environmental estrogens screening
Specific biomarkers, the estrogen that can evaluate Environmental Chemical Pollutants by vitellogenin level in detection milter body is living
Property.However, China also rarely has the research and development of marine environment POPs biological monitoring technology so far.
Pseudopleuronectes (Pseudopleuronectes yokohamae) are commonly called as husky plate, belong to Pleuronectiformes, Pangasiidae, Pseudopleuronectes category,
Long 120~the 240mm of general body, is cold warm nature bottom ocean fish, be distributed in north up to Korea, Tartar Strait and Hokkaido, Japan south with
And northern East China Sea is to the yellow Bohai Sea etc..Pseudopleuronectes are one of the Important Economic fish in the Huanghai Sea and the Bohai Sea, on China Shandong, Liaoning and other places
A large amount of cultivation.Pseudopleuronectes can buy acquisition on the market, and be easy to raise in laboratory, therefore this research selects the fish
Representative of the kind as aquatic products, establishes the quantitative measurement technology of its vitellogenin, helps to carry out China's immediate offshore area POPs
Biological monitoring work.
Summary of the invention
The technical problems to be solved by the present invention are: providing a kind of immunoblotting inspection for detecting Pseudopleuronectes fish vitellogenin
Test agent box, to meet the above-mentioned requirements of the prior art.
In order to solve the above technical problems, the technical solution adopted by the present invention is that:
A kind of immune-blotting method kit detecting Pseudopleuronectes fish vitellogenin provided by the invention, including a box
Body, 1 piece of 96 hole elisa Plates of blank, coating buffer, confining liquid, sample diluting liquid, cleaning solution, developing solution, terminator and horseradish peroxide
Goat-anti rabbit secondary antibody each 1 of compound enzyme label, it is characterised in that it is also equipped with Pseudopleuronectes fish lipovitellin sterling 1, mouse
The monoclonal antibody of anti-yellowing lid sole lipovitellin 1.
The preparation method of the ELISA kit of above-mentioned quantitative detection Pseudopleuronectes fish vitellogenin, feature includes following
Step: 1) Pseudopleuronectes fish lipovitellin sterling is prepared；2) the Pseudopleuronectes fish lipovitellin obtained using step 1) is pure
The monoclonal antibody of the product preparation anti-yellowing lid sole lipovitellin of mouse；3) the lipovitellin sterling 1 for obtaining step 1)
The monoclonal antibody 1 and coating buffer, confining liquid, sample for the anti-yellowing lid sole lipovitellin of mouse that branch, step 2) obtain
Dilution, cleaning solution, developing solution, goat-anti rabbit secondary antibody each 1 of terminate liquid and horseradish peroxidase-labeled be put into box, obtain
Detect the ELISA kit of Pseudopleuronectes fish vitellogenin.
The Pseudopleuronectes fish lipovitellin sterling the preparation method is as follows:
After dissecting Pseudopleuronectes, acquire mature fish-egg, be added 5 times of volumes ovum crude extract (20mmol/L Tris-HCl,
10mmol/L EDTA and 100mmol/L NaCl, pH 7.5), it is homogenized under condition of ice bath with glass homogenizer, 4 DEG C, 10000g
It is centrifuged 15min, after 0.45 μm of membrane filtration of supernatant, packing 1mL；Preliminary purification uses Sephacryl S-300 chromatographic column
1ml said extracted liquid is added, with 25mM Tris-HCl buffer (NaCl containing 0.07M and 1mM PMSF, pH in (2.0 × 80cm)
7.5) it at the uniform velocity elutes, elution flow rate 1ml/min, automatic fraction collector collects each eluting peak with every pipe 3ml；It is further purified
Using ion-exchange chromatography, above-mentioned elution fraction loading is taken, with Tris-HCl buffer (25mM, the pH of the NaCl containing 0.07M
7.5) it is eluted, flow velocity 1ml/min, collects eluent, as Pseudopleuronectes fish lipovitellin.
The monoclonal antibody of the anti-yellowing lid sole lipovitellin of the mouse the preparation method is as follows:
120 μ g Pseudopleuronectes fish lipovitellins are taken, Balb/C mouse is injected intraperitoneally after being mixed with Freund's complete adjuvant,
It is immunized again after two weeks, immunizing dose is 100 μ g, cannots be used up after full adjuvant mixes and is injected intraperitoneally；More than pressing every two weeks
Method is immunized, and the 5th time is booster immunization, and 3 days Pseudopleuronectes fish-eggs that adjuvant is not added in 100 μ g are yellow after the 4th injection
Rouge phosphoprotein is injected into mouse peritoneal and tail vein respectively；By hybridoma (2 × 106A/mL) it is injected into Mice Body, it is small
Mouse abdominal cavity has mentioned the atoleine for the last week injecting sterilizing；After mouse web portion expands, punctured with the syringe needle of 50mL syringe
Drainage obtains ascites, and purifying can be obtained monoclonal antibody from ascites.
The kit of above-mentioned quantitative detection Pseudopleuronectes fish vitellogenin ocean incretion upset chemical substance investigation with
Application in screening.
Beneficial effects of the present invention:
Kit of the invention sensitive, can be detected accurately, easily using the specific binding capacity between antigen-antibody
Vitellogenin in Pseudopleuronectes fish blood, in epidermal mucus, liver organization and hepatocyte cultures liquid, detection range 1.95
~250ng/mL provides an effective hand for the screening of endocrine disturbing chemical substance, detection and Ecological Environment Risk evaluation
Section.
Currently, both at home and abroad there is not yet about Pseudopleuronectes fish lipovitellin monoclonal antibody preparation report, the application
The preparation method for establishing Pseudopleuronectes fish lipovitellin monoclonal antibody for the first time, compared with polyclonal antibody, monoclonal is anti-
Body has higher antigen affinity and specificity, has higher susceptibility based on the detection method that monoclonal antibody is established.
The kit of this discovery, being capable of sensitive, easily quantitative detection Pseudopleuronectes using the specific binding capacity between antigen-antibody
The generation of vitellogenin is horizontal in vivo under the exposure of different pollutants for fish, for the environmental estrogens effect for evaluating China's immediate offshore area
Efficiently means should be provided.
Detailed description of the invention
Fig. 1 is kit of the invention to the testing result of Pseudopleuronectes fish vitellogenin sterling, and abscissa is Pseudopleuronectes
Fish log concentration value.
Fig. 2 is the testing result of kit of the invention to female/realgar lid sole blood plasma, and abscissa is sample extension rate
Logarithm.
Specific embodiment
Embodiment 1
The HP immunoblotting kit of preparation detection Pseudopleuronectes fish vitellogenin is divided into following steps:
(1) lipovitellin isolates and purifies
After dissecting Pseudopleuronectes, acquire mature fish-egg, be added 5 times of volumes ovum crude extract (20mmol/L Tris-HCl,
10mmol/L EDTA and 100mmol/L NaCl, pH 7.5), it is homogenized under condition of ice bath with glass homogenizer, 4 DEG C, 10000g
It is centrifuged 15min, after 0.45 μm of membrane filtration of supernatant, packing 1mL.Preliminary purification uses Sephacryl S-300 chromatographic column
1ml said extracted liquid is added, with 25mM Tris-HCl buffer (NaCl containing 0.07M and 1mM PMSF, pH in (2.0 × 80cm)
7.5) it at the uniform velocity elutes, elution flow rate 1ml/min, automatic fraction collector collects each eluting peak with every pipe 3ml.It is further purified
Using ion-exchange chromatography, above-mentioned elution fraction loading is taken, with Tris-HCl buffer (25mM, the pH of the NaCl containing 0.07M
7.5) it is eluted, flow velocity 1ml/min, collects eluent, as Pseudopleuronectes fish lipovitellin.
(2) preparation of Pseudopleuronectes fish lipovitellin monoclonal antibody
120 μ g Pseudopleuronectes fish lipovitellins are taken, Balb/C mouse is injected intraperitoneally after being mixed with Freund's complete adjuvant,
It is immunized again after two weeks, immunizing dose is 100 μ g, cannots be used up after full adjuvant mixes and is injected intraperitoneally.More than pressing every two weeks
Method is immunized, and the 5th time is booster immunization, and 3 days Pseudopleuronectes fish-eggs that adjuvant is not added in 100 μ g are yellow after the 4th injection
Rouge phosphoprotein is injected into mouse peritoneal and tail vein respectively.By hybridoma (2 × 106A/mL) it is injected into Mice Body, it is small
Mouse abdominal cavity has mentioned the atoleine for the last week injecting sterilizing.After mouse web portion expands, punctured with the syringe needle of 50mL syringe
Drainage obtains ascites, and purifying can be obtained monoclonal antibody from ascites.
(3) by the Pseudopleuronectes fish lipovitellin sterling 1 of preparation, the anti-yellowing lid sole lipovitellin monoclonal of mouse
Antibody 1, one piece of 96 hole elisa Plates of blank and coating buffer, confining liquid, sample diluting liquid, cleaning solution, developing solution, terminate liquid
In 1 loading box body of goat-anti rabbit secondary antibody of horseradish peroxidase-labeled, kit of the invention is constituted.Its concrete composition is such as
Under:
1) 1 piece of 96 hole elisa Plates of blank；
2) Pseudopleuronectes fish lipovitellin sterling 1 is diluted to required concentration with sample diluting liquid using preceding；
3) the anti-yellowing lid sole lipovitellin monoclonal antibody of mouse 1 uses the preceding volume that 1:5000 is pressed with coating buffer
Than dilution；
4) the sheep anti mouse secondary antibody of horseradish peroxidase-labeled 1 uses the preceding volume that 1:2000 is pressed with sample diluting liquid
Than dilution；
5) each 1 of coating buffer, cleaning solution, confining liquid, sample diluting liquid, developing solution, terminator,
The coating buffer is 9.6 carbonate buffer solution of 50mM pH: 1.59g Na2CO3, 2.93g NaHCO3, add distillation
Water is to 1000ml；
Cleaning solution is the 150mM pH7.4 phosphate buffer containing 0.05%Tween-20: 8.0g NaCl, 0.2g KCl,
2.9g Na2HPO412H2O, 0.2g KH2PO4,0.5ml Tween-20, add distilled water to 1000ml；
Confining liquid is the pH7.4 phosphate buffer containing 2%BSA: 0.2g BSA is dissolved in the phosphoric acid buffer of 10ml pH7.4
In liquid；
Sample diluting liquid is the 150mM phosphate buffer containing 0.05%Tween-20,1%BSA, pH 7.4:0.1g
BSA is dissolved in 10ml cleaning solution；
Developing solution is the TMB one pack system developing solution of Beijing Nuo Bo Rider Science and Technology Ltd. production；
Terminator is the H of 2M2SO4Aqueous solution.
The determination of sample diluting liquid composition
Sample diluting liquid is divided into 5 groups altogether, and the 1st group: the PBS of 0.15M；2nd group: the PBST (PBS of 0.15M, containing tween-
200.05%, pH 7.4)；3rd group: PBST (PBS of 0.15M contains tween-20 0.05%, pH 7.4)+1%BSA；4th
Group: PBST (PBS of 0.10M contains tween-20 0.05%, pH 7.4)+1%BSA；5th group: PBST (PBS of 0.20M, contains
Tween-20 0.05%, pH 7.4)+1%BSA, the Pseudopleuronectes fish-egg xanthan protein standard substance for being 20ng/ml with concentration carries out
It detects (method is referring to embodiment 2), determines that optimal sample diluting liquid, concrete outcome are as follows according to P/N value:
| P/N value | |
| Group 1 | 3.5 |
| Group 2 | 5.6 |
| Group 3 | 17.8 |
| Group 4 | 10.6 |
| Group 5 | 12.3 |
The preparation parameter of lipovitellin monoclonal antibody determines
In the purification process of lipovitellin monoclonal antibody, multiple parameters are adjusted, design parameter referring to
Following table, in addition to following table parameter, remaining step (2) during the preparation method is the same as that of Example 1.
Comparative example 5: purification step referring to patent CN103033630A qualitative detection of lipovitellin of cyprinid fish examination
(specific embodiment prepares the anti-goldfish ovum of mouse using the method for ammonium sulfate precipitation the 023rd section in agent box and its specification of application
Yellow phosphorus lipoprotein polyclonal antibody), other preparation methods are the same as 1 step of the embodiment of the present invention (2).
It is measured using potency of the indirect competitive to monoclonal antibody prepared by embodiment 1 and comparative example 1-4, indirectly
Competition law method is as follows: 1. being diluted the lipovitellin of purifying with 0.05mol/L carbonate coating buffer (pH 9.6)
To 2 μ g/ml, 100 μ L are added in every hole of 96 hole elisa Plates, 4 DEG C of coatings are overnight.2. washing: taking out ELISA Plate, restore to room
Temperature discards coating buffer, with PBST (PBS-0.5%Tween 20) board-washing 3 times, stands 1min every time, discards cleaning solution, absorbing water
ELISA Plate is patted dry on paper.3. closing: 200 μ l confining liquids (PBST-2%BSA), 37 DEG C of incubation 2h being added into every hole.4. plus
Enter antiserum: PBST board-washing 3 times, every hole be added with dilution (PBS-0.1%BSA) doubling dilution by antiserum (1:500~
1:128 000), 100 μ l, 37 DEG C of incubation 2h are added in every hole.5. ELIAS secondary antibody is added: PBST board-washing 5 times, 100 μ l are added in every hole
1:4000 times of diluted HRP label goat anti-rabbit igg (HRP-IgG), 37 DEG C of incubation 1h.6. colour developing: PBST board-washing 5 times, every hole adds
Enter 100 μ l of TMB one pack system developing solution, 37 DEG C are protected from light colour developing 10min, with 2mol/L H2SO450 μ l color development stoppings are used
Multiskan MK3 microplate reader measures 450nm absorbance.
As a result
The antiserum of the anti-yellowing lid sole lipovitellin of mouse carries out indirect ELISA detection, is computed the embodiment of the present invention 1
The anti-yellowing sero-fast potency of lid sole lipovitellin of the mouse of preparation is 1:32 000, and comparative example result see the table below.
| Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | |
| Potency | 1:4000 | 1:8000 | 1:8000 | 1:16000 | 1:8000 |
Embodiment 2
The HP immunoblotting kit of detection Pseudopleuronectes fish vitellogenin of the invention can be used for ocean incretion upsetization
Learn the detection of substance.Detection method includes the following steps:
1) with the Pseudopleuronectes fish lipovitellin in coating buffer dilution kit to 200ng/L, in 96 hole enzyme mark of blank
100 holes μ l/ are added in plate, 4 DEG C of coatings are incubated for 2 hours overnight or at 37 DEG C.Solution in hole is discarded, is washed 3 times.
2) confining liquid is added in 96 hole elisa Plates, 300 holes μ L/ are incubated for 1 hour at room temperature.Solution in hole is discarded, is washed
3 times.
3) addition is diluted with sample diluting liquid in 96 hole elisa Plates Pseudopleuronectes fish lipovitellin standard items, to
50 hole μ L/ of sample, and 1:5000 times of the sample diluting liquid diluted anti-yellowing lid sole egg fat phosphorus of mouse of 50 μ L is added in every hole
Protein antibodies are incubated for 2 hours at 37 DEG C.Solution in hole is discarded, is washed 5 times.
4) 1:2000 times of the sample diluting liquid diluted horseradish peroxidase-labeled goat-anti of addition in 96 hole elisa Plates
Rabbit secondary antibody, 100 holes μ L/ are incubated at room temperature 1 hour.Solution in hole is discarded, is washed 5 times.
5) developing solution of Fresh, 100 holes μ L/, in the reaction of 37 DEG C of room temperature dark place are added in 96 hole elisa Plates
10min。
6) add terminator, 50 holes μ L/ after apparent yellow to be presented.
7) with microplate reader measure 450nm wavelength under each hole light absorption value, measurement should after adding terminate liquid within 15 minutes into
Row.
8) calculate: using the logarithm of the concentration of reference substance as abscissa, OD value is ordinate, and standard is drawn on graph paper
Curve finds corresponding concentration by standard curve according to the OD value of sample；Multiplied by extension rate；Or with the concentration of reference substance with
OD value calculates the linear regression equation of standard curve, and the OD value of sample is substituted into equation, calculates sample concentration, multiplied by
With extension rate, the as actual concentrations of sample.Concrete outcome is referring to Fig. 1.
Detection of the kit of the present invention of embodiment 3 to female/realgar lid sole blood plasma
Using 17 beta estradiol of intramuscular injection (17 β-estradiol, E2) mode to induce Pseudopleuronectes fish to generate yolk former
Albumen.Injection takes blood after a week, carries out after gradually diluting to blood plasma；It is detected using the method for embodiment 2, it is specific to tie
Fruit referring to fig. 2
The determination of 4 minimum detection limit of embodiment
Taking Pseudopleuronectes fish-egg xanthan protein standard substance concentration is 10ng/ml, be gradually diluted to 5,2.5,1.25,0.625,
0.312 and 0.156ng/ml detects each concentration standards using the method for embodiment 2, and each concentration is arranged 2 again
Hole, blank well are added with the sample diluting liquid of standard item group equivalent as compareing, minimum when with the P/N value that measures greater than 2.1
Concentration is as minimum detection limit, and through detecting, the lowest detection of ELISA kit of the present invention is limited to 0.75ng/mL.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the art
Those of ordinary skill, within the essential scope of the present invention, the variations, modifications, additions or substitutions made all should belong to the present invention
Protection scope.
Claims (4)
1. a kind of HP immunoblotting kit for detecting Pseudopleuronectes fish vitellogenin, an including box body has blank 96 in box body
1 piece of hole elisa Plates, confining liquid, coating buffer, cleaning solution, sample diluting liquid, developing solution, terminate liquid and horseradish peroxidase-labeled
Goat-anti rabbit secondary antibody each 1, which is characterized in that the box body is also equipped with: Pseudopleuronectes fish lipovitellin sterling 1, mouse is anti-yellowing
The monoclonal antibody of lid sole lipovitellin 1.
2. kit as described in claim 1, which is characterized in that the preparation of the Pseudopleuronectes fish lipovitellin sterling
Method is as follows:
After dissecting Pseudopleuronectes, acquire mature fish-egg, be added 5 times of volumes ovum crude extract (20 mmol/L Tris-HCl, 10
Mmol/L EDTA and 100 mmol/L NaCl, pH 7.5), it is homogenized under condition of ice bath with glass homogenizer, 4 DEG C, 10000
G is centrifuged 15 min, after 0.45 μm of membrane filtration of supernatant, 1 mL of packing；Preliminary purification uses Sephacryl S-300 layers
It analyses column (2.0 × 80 cm), 1 ml said extracted liquid is added, (contain 0.07 M NaCl and 1 with 25 mM Tris-HCl buffers
MM PMSF, pH 7.5) at the uniform velocity elute, elution flow rate is 1 ml/min, and automatic fraction collector collects each elution with every 3 ml of pipe
Peak；It is further purified using ion-exchange chromatography, takes above-mentioned elution fraction loading, it is slow with the Tris-HCl containing 0.07 M NaCl
Fliud flushing (25 mM, pH 7.5) is eluted, and flow velocity is 1 ml/min, collects eluent, as Pseudopleuronectes fish egg fat phosphorus egg
It is white.
3. kit as described in claim 1, which is characterized in that the Dan Ke of the anti-yellowing lid sole lipovitellin of the mouse
Grand antibody the preparation method is as follows:
120 μ g Pseudopleuronectes fish lipovitellins are taken, Balb/C mouse are injected intraperitoneally after being mixed with Freund's complete adjuvant, two
It is immunized again after week, immunizing dose is 100 μ g, cannots be used up after full adjuvant mixes and is injected intraperitoneally；Every two weeks by with top
Method is immunized, and the 5th time is booster immunization, and 100 μ g are not added to the Pseudopleuronectes fish egg fat of adjuvant for 3 days after the 4th injection
Phosphoprotein is injected into mouse peritoneal and tail vein respectively；By hybridoma (2 × 106A/mL) it is injected into Mice Body, mouse
Abdominal cavity has mentioned the atoleine for the last week injecting sterilizing；After mouse web portion expands, drawn with the syringe needle puncture of 50 mL syringes
Stream obtains ascites, and purifying can be obtained monoclonal antibody from ascites.
4. detection Pseudopleuronectes fish-egg xanthan protein immunoblot kit described in claim 1 is in marine environment estrogens object
Matter screening and the application in the research of endocrine disturbing chemical substance.
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| CN110646335A (en) * | 2019-09-29 | 2020-01-03 | 广东工业大学 | Sealing liquid and application thereof |
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