CN109884312A - A kind of colloid gold test paper detecting Bastard halibut vitellogenin - Google Patents
A kind of colloid gold test paper detecting Bastard halibut vitellogenin Download PDFInfo
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- CN109884312A CN109884312A CN201910070321.1A CN201910070321A CN109884312A CN 109884312 A CN109884312 A CN 109884312A CN 201910070321 A CN201910070321 A CN 201910070321A CN 109884312 A CN109884312 A CN 109884312A
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- bastard halibut
- lipovitellin
- vitellogenin
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Abstract
A kind of colloid gold test paper detecting Bastard halibut vitellogenin.Lipovitellin is rapidly purified out from Bastard halibut ovary homogenate by simple water sedimentation method, Bastard halibut lipovitellin polyclonal antibody and Bastard halibut lipovitellin monoclonal antibody are prepared, and will be on 20 nm colloid gold labels to the monoclonal antibody of purifying of preparation；The monoclonal antibody of colloid gold label is sprayed in gold-labelled pad, Bastard halibut lipovitellin polyclonal antibody is sprayed on nitrocellulose filter, nitrocellulose filter, gold conjugation pad, sample pad, water absorption pad are successively pasted in PVC board, the strip for being 4 mm at width by assembled PVC board cutting machine longitudinal shear as detects the colloid gold test paper of Bastard halibut vitellogenin.The test paper can complete the detection of Bastard halibut vitellogenin within 10 min, and marine environment estrogen is quickly detected for field condition and provides important tool.
Description
Technical field
The invention belongs to environmental testings, are related to a kind of colloid gold test paper and its system for detecting Bastard halibut vitellogenin
Preparation Method.
Background technique
Environmental estrogens are as a kind of compound closely related with human lives, with not degradable, residual life is long, close
The features such as lipid, can interfere the generation, release and physiological function of organism inner estrogen, thus growth, development to organism
Have an adverse effect with fertility, or even will cause breast cancer and the raising of testis cancer morbidity.Environmental estrogens can pass through dirt
The approach such as water discharge, atmospheric sedimentation and rain drop erosion are finally aggregated into ocean, and estrogen in marine environment is caused to pollute increasingly
Seriously, estradiol such as in China's coastal seawater, female alkynol, the common estrogen of bisphenol-A concentration be respectively 45.7 ng/L,
3.99 ng/L,3920 ng/L.These pollutants can cause to seriously endanger under the level of ng/L to organism, such as 1ng/
The female alkynol exposure of L will lead to male bottom Medaka Gonad dysplasia, risk of feminizing occurs.
A kind of biomarker of the vitellogenin (vitellogenin, Vtg) as environmental estrogens of detection extensively,
It can accurately indicate estrogen-like contaminant to the disturbing effect of aquatic animal.Vitellogenin is under the induction of exogenous estrogen
It is synthesized by liver, through blood transportation into ovary, the components such as lipovitellin is decomposed into ovary.Detection yolk egg at present
White original mainly has the methods of enzyme-linked immunosorbent assay, protein blot experiment and chromatography.These means of testing mainly exist
It is carried out in laboratory, need to possess more complex experiment condition and has good experimental skill.Swash as people are female to environment
The deep understanding of element interference, estrogen will develop into the conventional index of pollution detection, and just there is an urgent need to develop a kind of behaviour for this
Make common detection methods that are simple, cheap, can adapt to field condition detection.Immune colloid gold test paper is to show with colloidal gold
Track agent carries out the detection of antigen, and it is simple, quick, sensitive, special, good without supplementary instrument equipment, stability to have the characteristics that, right
It is of great significance in the environmental estrogens biomarker detection technique for developing easy to carry, sensitive, easy.However, the party
Application of the method in the detection of marine environment estrogen is also in blank.For the pollution situation of accurate evaluation China coastal seas estrogen,
Need the vitellogenin colloidal gold test that exploitation is directed to China ocean fingerling.
Bastard halibut is under the jurisdiction of Pleuronectiformes, lefteye flounder section, and Paralichthys are omnivorousness benthic fishes, is distributed mainly on the Huang of China
The Bohai Sea, the East Sea and Korea, the Japanese stretch of coastal water.Currently, Bastard halibut China, South Korea, Japan it is coastal have cultivation, be important
Marine culture and stock enhancement fingerling.In the case where marine environmental pollution is on the rise, more and more scholars start flounder flounder class to make
Be marine pollutant to the experimental model of marine fishes Pollution Study, by pollutant is metabolized in flounder flounder class body, is distributed with
And on the research that reproductive development etc. influences, poisonous effect of the Lai Fanying pollutant to marine organisms.Therefore using brown
The biomarker Fast Detection Technique of the vitellogenin exploitation marine environment estrogen of lefteye flounder is of great significance.
Summary of the invention
The purpose of the present invention is to provide a kind of colloid gold test papers for detecting Bastard halibut vitellogenin, to meet existing skill
The above-mentioned requirements of art.
A kind of colloidal gold strip detecting Bastard halibut vitellogenin, including PVC offset plate, PVC offset plate upper surface are equipped with
Sample pad, water absorption pad, nitrocellulose filter and bonding pad；
It is characterized in that the bonding pad has been coated with the Bastard halibut lipovitellin monoclonal antibody of golden label, the nitre
Acid cellulose film has been coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti-mouse igg antibody.
The colloidal gold strip of the detection Bastard halibut vitellogenin, it is characterised in that described has been coated with colloidal gold
The bonding pad of the Bastard halibut lipovitellin monoclonal antibody of label is to prepare using the following method:
It weighs after Bastard halibut ovary is taken out, the 0.01 M PBS buffer solution that 4 DEG C of 5 times of volumes pre-coolings are added mixes, in ice bath item
It is homogenized under part using glass homogenizer, 5000 g are centrifuged 5 min and 10 times of bodies are added after 0.45 μm of membrane filtration of supernatant
The ultrapure water of 4 DEG C of pre-coolings of product, rocks after ten minutes on shaking table, and 5000 g are centrifuged 10 min, takes supernatant, obtains Bastard halibut ovum
Yellow rouge phosphoprotein sterling；It takes Bastard halibut lipovitellin sterling that Balb/c mouse is immunized, takes spleen cell, with SP2/0 marrow
Oncocyte fusion, obtains the positive hybridoma cell of specificity after being subcloned three times；By 2 × 106The positive hybridoma of a/mL
In cell intraperitoneal injection to Mice Body, ascites is taken after a week, and 3000 g are centrifuged 10 min, collect supernatant, survey using indirect ELISA
Determine the potency of ascites；It is purified from ascites supernatant using Protein G affinity column and obtains monoclonal antibody；Take the 20 of 1 mL
The K of 0.2 M of 1 μ L is added in nm colloidal gold2CO3, while 16 μ g monoclonal antibodies are added, 30 min are reacted, add 10%
110 μ L of BSA closes 30 min, and 3000 r/min are centrifuged 10 min, and after abandoning precipitating, 11000 r/min are centrifuged 30 min, discards
Supernatant is re-dissolved precipitating with the PBS (PH=7.4, the sucrose containing 5 %, the BSA of 1 %) of 0.01 M of 100u L；Take one
Item grows 10 cm, and above-mentioned 400 μ l immune colloid gold is equably coated in the glass fibre membrane by the glass fibre membrane of wide 0.5 cm
On, it after 37 DEG C of dryings, plastic packaging bag sealing, is saved in 4 DEG C, that is, obtains the Bastard halibut egg fat phosphorus egg for being coated with colloid gold label
The bonding pad of white monoclonal antibody.
The colloidal gold strip of the detection Bastard halibut vitellogenin, it is characterised in that described has been coated with Bastard halibut
The nitrocellulose filter of lipovitellin polyclonal antibody and sheep anti-mouse igg antibody is to prepare using the following method:
The nitrocellulose filter of 30 cm is attached in PVC board, will by conventional method Bastard halibut lipovitellin obtained it is more
After clonal antibody is diluted to 0.8 mg/mL, detection line position on even application to nitrocellulose filter, by commercially available sheep anti mouse
IgG antibody is diluted to 6 mg/mL, and the Quality Control line position on even application to nitrocellulose filter is dried overnight, then in 37 DEG C
After the sealing of plastic packaging bag, saved backup in 4 DEG C；It obtains and has been coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti mouse
The nitrocellulose filter of IgG antibody.
The colloid gold test paper of the detection Bastard halibut vitellogenin, it is quick at marine environment estrogenic chemicals scene
Application in detection.
Advantages of the present invention
Since the vitellogenin of fish is easy degradation during isolating and purifying and storing, to avoid the degradation of Vtg to detection
As a result deviation caused by, the present invention is using more stable and lipovitellin with Vtg with identical immunogenicity as fixed
The standard items of amount.In addition, the present invention improves purifying Bastard halibut lipovitellin scheme for the first time, can be purified within 1 h
Bastard halibut lipovitellin out greatly reduces workload, and cheaper with the equipment price used, can be in laboratory
In carry out the work.The present invention is prepared for Bastard halibut egg fat phosphorus egg monoclonal antibody and polyclonal antibody for the first time, improves simultaneously
The preparation method of monoclonal antibody improves the effect of the fusion rate of hybridoma preparation process and the monoclonal antibody of preparation
Valence, while using two kinds of antibody of preparation, a kind of colloidal gold immunochromatographimethod technology is established for the first time, is quickly detected for field brown
Lefteye flounder vitellogenin.
Colloid gold test paper prepared by the present invention is easy to operate, easy to carry, testing cost is cheap, is not necessarily to supplementary instrument equipment
It can complete to detect accordingly, be particularly suitable for field and quickly detect vitellogenin in fish body；The present invention can be sensitive, quasi-
Really, Bastard halibut vitellogenin is easily detected, is screening, detection and the Ecological Environment Risk evaluation of environmental estrogens substance
Provide effective means.
Detailed description of the invention:
Fig. 1 is the optimization figure of the test strip optimum protein labelled amount of Bastard halibut vitellogenin.
Fig. 2 is the optimization figure of the test strip optimum mark PH of Bastard halibut vitellogenin.
Fig. 3 is the optimization figure of the best T line of test strip of Bastard halibut vitellogenin.
Fig. 4 is the optimization figure of the best C line of test strip of Bastard halibut vitellogenin.
Fig. 5 is actually detected figure of the test strips for various concentration Bastard halibut vitellogenin.
Fig. 6 is the two dimensional plot that test strips detect various concentration Bastard halibut vitellogenin.
Fig. 7 is that the test strip actual sample of Bastard halibut vitellogenin detects figure.
Specific embodiment:
A kind of colloidal gold strip detecting Bastard halibut vitellogenin, including PVC offset plate, PVC offset plate upper surface are equipped with sample
Pad, water absorption pad, nitrocellulose filter and bonding pad；
It is characterized in that the bonding pad has been coated with the Bastard halibut lipovitellin monoclonal antibody of golden label, the nitre
Acid cellulose film has been coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti-mouse igg antibody.
In the colloid gold test paper of above-mentioned detection Bastard halibut vitellogenin, the Bastard halibut egg fat phosphorus egg of the gold label
White monoclonal antibody, the nitrocellulose filter for being coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti-mouse igg antibody
It is to prepare in the following manner:
(1) Bastard halibut lipovitellin sterling is prepared；
(2) Bastard halibut lipovitellin Anti-TNF-α is prepared using the Bastard halibut lipovitellin sterling that step (1) obtains
Body；
(3) Bastard halibut lipovitellin monoclonal is prepared using the Bastard halibut lipovitellin sterling that step (1) obtains to resist
Body；
(4) the Bastard halibut lipovitellin monoclonal antibody of the Antibody preparation colloid gold label obtained using step (3)；
(5) the Bastard halibut lipovitellin monoclonal antibody preparation obtained using step (4) has been coated with colloid gold label Dan Ke
The bonding pad of grand antibody；
(6) the Bastard halibut lipovitellin polyclonal antibody preparation coating polyclonal antibody and goat-anti obtained using step (2)
The nitrocellulose filter of mouse IgG antibody；
(7) nitrocellulose filter, gold conjugation pad, processed glass fibers successively the assembling of test strips: are pasted in PVC board
Film, blotting paper are tieed up, the strip for being 5 mm at width by assembled PVC board cutting machine longitudinal shear, as Bastard halibut yolk
The Rapid detection test strip of rouge phosphoprotein.
Below by specific embodiment and in conjunction with attached drawing, the present invention is further elaborated.
Embodiment:
A kind of colloidal gold strip detecting Bastard halibut vitellogenin, including bonding pad, nitrocellulose filter, sample pad, suction
Water cushion, PVC offset plate and plastic mould.
(1) it the preparation of Bastard halibut lipovitellin sterling: weighs after Bastard halibut ovary is taken out, 5 times of volumes 4 is added
DEG C pre-cooling 0.01 M PBS buffer solution mix, be homogenized under condition of ice bath using glass homogenizer, 5000g5 min are centrifuged,
After 0.45 μm of membrane filtration of supernatant, the ultrapure water of 4 DEG C of 10 times of volumes pre-coolings is added, is rocked on shaking table after ten minutes,
5000 g are centrifuged 10 min, take supernatant, i.e. acquisition Bastard halibut lipovitellin sterling；
(2) preparation of Bastard halibut lipovitellin monoclonal antibody: by 0.1 mL contain 70 μ g lipovitellin and
It is injected into mouse peritoneal after isometric not formula Freund's complete adjuvant emulsification, after two weeks with same dose of lipovitellin and Freund
Booster immunization is carried out to Balb/c mouse after Freund's incomplete adjuvant is fully emulsified, tail vein injection lipovitellin carries out after a week
Booster immunization puts to death mouse and mouse spleen is taken to carry out cell fusion after three days；Fusion the previous day, after normal mouse is put to death, nothing
Bacterium takes Peritoneal Cells of Mice, is diluted to 2 × 10 with complete medium after centrifugation5After a/mL, it is thin that 96 holes are laid on by 100 holes μ L/
Born of the same parents' culture plate is as feeder cells；The spleen cell for taking mouse after booster immunization will under the action of fusion agent PEG 4000
Spleen cell carries out cell fusion in the ratio of 5:1 with the myeloma cell (SP2/0) in logarithmic growth phase；Fused production
Object is used containing after 20% FBS dilution, is laid in 96 orifice plates by 100 holes μ L/, second day to 96 orifice plates containing fused cell
The complete medium of 10 × HAT of 50 μ L is added.Culture filters out positive hole after a week, using indirect ELISA, and utilization has
Dilution method is limited to be subcloned three times the hybridoma of secretion Bastard halibut lipovitellin antibody；The liquid that 500 μ L are sterilized
Paraffin is injected into mouse peritoneal, week post injection positive hybridoma cell (2 × 10 in advance6A/mL).After about 7 days, with No. 16
Pin puncture drainage takes ascites, and 3000 g are centrifuged 10 min, collects supernatant, utilizes the potency of indirect ELISA measurement ascites.It utilizes
Protein G affinity column purifies from ascites supernatant and obtains monoclonal antibody (mAb).The antibody of purifying is dialysed through ultrapure water
After 24 h, that is, obtain Bastard halibut lipovitellin monoclonal antibody；
(3) it the preparation of the anti-Bastard halibut lipovitellin monoclonal antibody of the mouse of colloid gold label: is restored using trisodium citrate
Method prepares the colloidal gold that partial size is 20 nm or so；
(4) determination of colloidal gold labeled monoclonal antibody optimum protein amount: taking the centrifuge tube of 8 1.5 mL, respectively label 1,2,3,4,5,
6, the colloidal gold of the above-mentioned preparation of 1 mL is added in 7, No. 8 pipes, every pipe, and each pipe is separately added into 1,2,4,8,16,32, the 64 brown teeth of μ g
Flounder lipovitellin monoclonal antibody；The K of 10 μ L (excess), 0.2 M is added in each pipe2CO3Solution mixes, is placed at room temperature for 30 min
Afterwards；Each pipe is separately added into 10% 100 μ L of NaCl solution again, mixes, and observation is as a result, the variation by color is sentenced after standing 2 h
Whether have coagulation phenomenon, and then come the optimum protein amount that determines colloid gold label if determining colloidal gold.Bastard halibut yolk of the invention is former
The optimization figure of the test strip optimum protein labelled amount of albumen is as shown in Figure 1.
(5) determination of the best pH value of colloid gold label monoclonal antibody: taking the centrifuge tube of 6 1.5 mL, respectively label 1,
2,3,4,5, No. 6 pipes, are added 1 mL colloidal gold and 20 μ g Bastard halibut lipovitellin monoclonal antibodies, are then added 0.2
The K of M2CO31,2,4,6,8,10 μ L of solution is mixed, and after standing 30 min, is separately added into 10% 100 μ L of NaCl solution, is mixed
It is even.It is observed after standing 2 h as a result, the variation by color determines whether colloidal gold has coagulation phenomenon, then by the colloid of label
Gold spraying determines the best pH value of colloid gold label in gold-labelled pad after colour developing.The inspection of Bastard halibut vitellogenin of the invention
The optimization figure for testing paper slip optimum mark PH is as shown in Figure 2.
(6) prepared by colloidal gold-monoclonal antibody compound: taking 1 mL colloidal gold, the K of 1 μ L, 0.2 M is added2CO3,
20 μ g monoclonal antibodies are added simultaneously, react 30 min, add 10% 110 μ l of BSA closing 30 min, 3000 r/min
Be centrifuged 10 min, after abandoning precipitating, 11000 r/min are centrifuged 10 min, discard supernatant, with the redissolution liquid of 100ul (0.01 M's
PBS (PH=7.4, containing 5% sucrose, 1% BSA), precipitating is re-dissolved, 4 DEG C of refrigerators is placed in and saves backup.
(7) preparation of gold conjugation pad: take it is one long take 10 cm one long, the glass fibre membrane of wide about 0.5 cm,
Above-mentioned immune colloid gold is equably coated on the glass fibre membrane, it is standby in 4 DEG C of preservations after 37 DEG C of dryings, plastic packaging bag sealing
With.
(8) sheep anti-mouse igg antibody of 8 mg/mL the best diluted concentration of T line: is sprayed at nitrocellulose filter as C
Bastard halibut lipovitellin polyclonal antibody is diluted to 0.05,0.1,0.2,0.4,0.6,0.8 mg/mL and sprayed respectively by line
In nitrocellulose filter as T line, best T line concentration is observed after colour developing.The detection of Bastard halibut vitellogenin of the invention
The optimization figure of the best T line of test strips is as shown in Figure 3.
(9) the Bastard halibut lipovitellin polyclonal antibody of 0.8 mg/mL the best diluted concentration of C line: is sprayed at nitre
Sheep anti-mouse igg antibody is diluted to 0.5,1,2,4,6,8 mg/mL and is sprayed at cellulose nitrate respectively by acid cellulose film as T line
Plain film observes best C line concentration as C line after colour developing.The best C of test strip of Bastard halibut vitellogenin of the invention
The optimization figure of line is as shown in Figure 4.
(10) preparation process of nitrocellulose filter: the nitrocellulose filter (NC film) of 30 cm is attached in PVC board, brown
Lefteye flounder lipovitellin polyclonal antibody and sheep anti-mouse igg are diluted to suitable concentration with the PBS of the M of pH=7.2 0.01, so
Afterwards by the two even application to NC film, respectively as detection line (T) and nature controlling line (C), in 37 DEG C of dry 2 h, taking-up is placed in
In drying cupboard overnight, it is then saved backup with after the sealing of plastic packaging bag in 4 DEG C.
(11) nitrocellulose filter, gold conjugation pad, sample pad, water absorption pad are successively pasted in PVC board, will be assembled
The longitudinal shear of PVC board cutting machine be 4 mm at width strip, the as quick detection test paper of Bastard halibut vitellogenin
Item.Test strips are as shown in Figure 5,6 for the actually detected figure and standard curve of various concentration Bastard halibut vitellogenin.
A kind of colloidal gold strip of detection Bastard halibut lipovitellin of the invention can be used for marine environment estrogen
Quick detection, sample to be tested is added dropwise at the sample pad of colloidal gold strip by when use, observe chromogenic reaction after 10 min,
The actual sample detection case of the test strip of Bastard halibut vitellogenin of the invention is as shown in Figure 7.
Claims (4)
1. a kind of colloidal gold strip for detecting Bastard halibut vitellogenin, including PVC offset plate, PVC offset plate are covered with sample on surface
Product pad, water absorption pad, nitrocellulose filter and bonding pad；
It is characterized in that the bonding pad has been coated with the Bastard halibut lipovitellin monoclonal antibody of golden label, the nitre
Acid cellulose film has been coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti-mouse igg antibody.
2. the colloidal gold strip of detection Bastard halibut vitellogenin according to claim 1, it is characterised in that described
The bonding pad for being coated with the Bastard halibut lipovitellin monoclonal antibody of colloid gold label is to prepare using the following method:
It weighs after Bastard halibut ovary is taken out, the 0.01 M PBS buffer solution that 4 DEG C of 5 times of volumes pre-coolings are added mixes, in ice bath item
It is homogenized under part using glass homogenizer, 5000 g are centrifuged 5 min and 10 times of bodies are added after 0.45 μm of membrane filtration of supernatant
The ultrapure water of 4 DEG C of pre-coolings of product, rocks after ten minutes on shaking table, and 5000 g are centrifuged 10 min, takes supernatant, obtains Bastard halibut ovum
Yellow rouge phosphoprotein sterling；It takes Bastard halibut lipovitellin sterling that Balb/c mouse is immunized, takes spleen cell, with SP2/0 marrow
Oncocyte fusion, obtains the positive hybridoma cell of specificity after being subcloned three times；By 2 × 106The positive hybridoma of a/mL
In cell intraperitoneal injection to Mice Body, ascites is taken after a week, and 3000 g are centrifuged 10 min, collect supernatant, survey using indirect ELISA
Determine the potency of ascites；It is purified from ascites supernatant using Protein G affinity column and obtains monoclonal antibody；Take the 20 of 1 mL
The K of 0.2 M of 1 μ L is added in nm colloidal gold2CO3, while 16 μ g monoclonal antibodies are added, 30 min are reacted, add 10%
110 μ L of BSA closes 30 min, and 3000 r/min are centrifuged 10 min, and after abandoning precipitating, 11000 r/min are centrifuged 30 min, discards
Supernatant is re-dissolved precipitating with the PBS (PH=7.4, the sucrose containing 5 %, the BSA of 1 %) of 0.01 M of 100u L；Take one
Item grows 10 cm, and above-mentioned 400 μ l immune colloid gold is equably coated in the glass fibre membrane by the glass fibre membrane of wide 0.5 cm
On, it after 37 DEG C of dryings, plastic packaging bag sealing, is saved in 4 DEG C, that is, obtains the Bastard halibut egg fat phosphorus egg for being coated with colloid gold label
The bonding pad of white monoclonal antibody.
3. the colloidal gold strip of detection Bastard halibut vitellogenin according to claim 1, it is characterised in that described
The nitrocellulose filter for being coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti-mouse igg antibody is to make using the following method
It is standby:
The nitrocellulose filter of 30 cm is attached in PVC board, will by conventional method Bastard halibut lipovitellin obtained it is more
After clonal antibody is diluted to 0.8 mg/mL, detection line position on even application to nitrocellulose filter, by commercially available sheep anti mouse
IgG antibody is diluted to 6 mg/mL, and the Quality Control line position on even application to nitrocellulose filter is dried overnight, then in 37 DEG C
It after the sealing of plastic packaging bag, is saved backup in 4 DEG C, that is, obtains and be coated with Bastard halibut lipovitellin polyclonal antibody and sheep anti mouse
The nitrocellulose filter of IgG antibody.
4. the colloid gold test paper of detection Bastard halibut vitellogenin described in claim 1, in marine environment estrogenic chemicals
Application in field quick detection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110726847A (en) * | 2019-11-01 | 2020-01-24 | 厦门大学 | Method for detecting estrogen pollution of water body based on recombinant bostrichthys sinensis vitellogenin and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1451965A (en) * | 2002-04-18 | 2003-10-29 | 中国科学院水生生物研究所 | Method for preparing quick estrogenoid toxicity detecting test piece |
| KR20040027222A (en) * | 2002-09-27 | 2004-04-01 | 바이오돔(주) | Biomarker Reagent for the detection of Endocrine Disruptors |
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2019
- 2019-01-25 CN CN201910070321.1A patent/CN109884312A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1451965A (en) * | 2002-04-18 | 2003-10-29 | 中国科学院水生生物研究所 | Method for preparing quick estrogenoid toxicity detecting test piece |
| KR20040027222A (en) * | 2002-09-27 | 2004-04-01 | 바이오돔(주) | Biomarker Reagent for the detection of Endocrine Disruptors |
Non-Patent Citations (4)
| Title |
|---|
| JUN WANG, ET AL.: "Development of a lipovitellin-based goldfish (Carassius auratus) vitellogenin ELISA for detection of environmental estrogens.", 《CHEMOSPHERE》 * |
| SAMUEL G.PRAKASH VINCENT, ET AL.: "Development of Vitellogenin-ELISA, an In Vivo Bioassay, and Identification of Two Vitellogenesis-Inhibiting Hormones of the Tiger Shrimp Penaeus monodon.", 《MARINE BIOTECHNOLOGY》 * |
| 张振忠，等: "鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立。", 《中国水产科学》 * |
| 王军: "斑马鱼卵黄原蛋白单/多克隆抗体的制备及其在环境雌激素检测中的应用。", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110726847A (en) * | 2019-11-01 | 2020-01-24 | 厦门大学 | Method for detecting estrogen pollution of water body based on recombinant bostrichthys sinensis vitellogenin and application |
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