CN107271684A - A kind of kit quantitatively detected for seawater medaka vitellogenin, preparation method and application - Google Patents
A kind of kit quantitatively detected for seawater medaka vitellogenin, preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of kit quantitatively detected for seawater medaka vitellogenin, preparation method and application, belongs to environmental monitoring.Kit includes 1 piece of hole elisa Plates of blank 96,1 seawater medaka vitellogenin standard items, 1 rabbit-anti seawater medaka vitellogenin antibody, the rabbit-anti seawater medaka vitellogenin antibody of 1 horseradish peroxidase-labeled, coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 of terminate liquid.This kit can be in the sample such as quick, sensitive, high specific quantitative detection seawater medaka blood plasma, tissue homogenate, epidermal mucus vitellogenin, minimum detection limit reaches 60ng/mL, and the biological monitoring that can be polluted for marine environment estrogen provides technological means；In addition, compared to other detection methods, the kit also has easy to operate, quick advantage.
Description
Technical field
The invention belongs to environmental monitoring, it is related to a kind of reagent quantitatively detected for seawater medaka vitellogenin
Box, can be used in marine environment estrogen pollution monitoring.
Background technology
Environmental estrogens are the various environmental contaminants of a type, can interfere with the synthesis of biological hormone in vivo, release,
Transport, metabolism, with reference to etc. a series of biological processes, so as to upset the normal hormonal level and internal system machine in organism
Can, and further produce adverse effect to ecosystem health, such as the influence reproductive process of wild animal, population sex ratio and
Scale etc..In recent years, the pollution problem of marine environment estrogen causes increasing concern, in Japan's Tokyo bay, Canada
The marine site such as St. John's port and Spain's Bay of Biscay is found that different degrees of marine environment estrogen pollution.In China
It is again seen that similar situation.For example, being up to by the wild mullet hermaphroditic incidence of Liaodong Wan of Effects of environmental estrogens
More than 50%.At present, environmental estrogens have become one of key factor of threat marine environment health.
Level of pollution to environmental estrogens accurately monitor and assessment is the necessary ways for realizing its contamination control.
Compared to traditional chemical monitoring, biological monitoring can intuitively reflect the comprehensive toxicity effect of environmental estrogens, be even more ideal
Environmental estrogens pollution monitoring technological means.Wherein, fish vitellogenin (Vitellogenin, Vtg) Induction experiments
It is the most commonly used environmental estrogens monitoring method.Vtg is the precursor of livetin, in the thorn of the beta estradiol of period of yolk formation 17
Under swashing, synthesized by liver and secreted, ovary is transported to by blood circulation, be used as the nutrient source of embryonic development.In normal conditions
Under, the raun that Vtg generates the phase by yolk largely synthesizes, and milter can not synthesize with juvenile fish because lacking estrogen, but its liver
With ERs, in the presence of exogenous hormone, it can also be induced synthesis and secretion Vtg.Therefore, milter or juvenile fish body
Interior Vtg levels are the ideal indicators for indicating environmental estrogens pollution.
At present, the pattern fish for having set up test of maturity method are freshwater fish, such as zebra fish, fathead minnow, rare
Minnow crucian carps etc..The difference of briny environment and fresh water environment cause ecological toxicology characteristic of the environmental estrogens in marine environment with it is light
Water environment is significantly different, and these fresh water bioorganisms are difficult to the environmental estrogens class material being directly used in monitoring marine environment.
Seawater medaka (Oryzias melastigma), also known as the blue or green Medaka of stain, originate in Pakistan, India, Burma and Thailand edge
Sea and freshwater, with it is small, to react sensitive, easily recognizable male and female, salinity adaptation scope to environmental pollutants wide etc. excellent
Point, is that emerging marine ecology toxicological profile is biological.The seawater medaka Vtg quantitative detecting methods of standardization are developed, will be had
Help the monitoring and control of marine environment estrogen pollution.Seawater medaka Vtg homologous antibodies are used however, not yet setting up at present
Quantitative detecting method.Although can also detect seawater medaka Vtg using heterologous antibody, homologous antibody is compared, it is heterologous anti-
The detection sensitivity and specificity of body are relatively low, constrain application of the seawater medaka in marine environment estrogen pollution monitoring.
Therefore, need the homologous antibody for preparing seawater medaka Vtg badly, and set up corresponding quantitative detecting method and commercial kit.
The content of the invention
In view of the shortcomings of the prior art, the present invention provides a kind of examination quantitatively detected for seawater medaka vitellogenin
Agent box, preparation method and application.
The technical scheme is that：
A kind of kit quantitatively detected for seawater medaka vitellogenin, the kit includes 1 box body, box body
Built with：1 piece of hole elisa Plates of blank 96；1 seawater medaka vitellogenin standard items, is diluted using preceding with sample diluting liquid
To required concentration；1 rabbit-anti seawater medaka vitellogenin antibody, 5 μ g/mL are diluted to coating buffer using preceding；1 horseradish
The rabbit-anti seawater medaka vitellogenin antibody of peroxidase labelling, using it is preceding with sample diluting liquid press 1：2500 ratio
Dilution；Coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 bottle of terminate liquid.
Described coating buffer is the carbonate buffer solutions of 50mM pH 9.6；Described confining liquid is the pH 7.4 containing 2%BSA
Phosphate buffer；Described sample diluting liquid is the phosphate buffer containing 0.05%Tween-20 and 1%BSA；It is described
Cleaning solution be the 150mM pH 7.4 containing 0.05%Tween-20 phosphate buffer；Described nitrite ion is TMB single groups
Divide nitrite ion；Described terminate liquid is 2M H2SO4The aqueous solution.
The preparation method of the above-mentioned kit quantitatively detected for seawater medaka vitellogenin, mainly prepares seawater blue or green
The rabbit-anti sea of Medaka fish-egg xanthan protein standard substance, rabbit-anti seawater medaka vitellogenin antibody and horseradish peroxidase-labeled
Water medaka vitellogenin antibody, comprises the following steps：
The preparation method of described seawater medaka vitellogenin standard items, comprises the following steps：
1) the method induction seawater medaka synthesis vitellogenin of 17 beta estradiols is exposed using water body, wherein, 17 β-
The exposure concentrations of estradiol are 50~200 μ g/L.Seawater medaka adult fish is placed in 5L glass aquarium, each bath of glass
10 tail seawater medakas are put into race's case；Water is all changed daily, and rejoins 17 beta estradiols of respective amount, keeps exposure dense
Degree is constant；Sooner or later feeding bait, keeps 25 DEG C of water temperature, natural lighting.
2) the seawater medaka after exposing 10 days is taken, is weighed, the ice-cold homogenate buffering of seawater 3 times of quality of medaka is added
Liquid, ice bath homogenate, homogenate low-temperature centrifugation 10 minutes collects supernatant.Described homogenate buffer is 25mM Tris, is included
0.07M NaCl and 0.5TIU/mL Aprotinins, pH 7.6.
3) supernatant being collected into is used for gel permeation chromatography, with 25mM Tri-HCl homogenate buffers with 1mL/min
Flow velocity swash of wave chromatographic column, collect main eluting peak, wherein, 25mM Tri-HCl homogenate buffers include 0.07M NaCl and
0.5TIU/mL Aprotinins, pH 7.6.Eluent is added into anion exchange chromatography again, with respectively contain 0.07,0.1,0.2
Continue to elute with 1mL/min flow velocity with 1M NaCl 25mM Tris-HCl homogenate buffers, each gradient washes 60min,
0.2M eluting peaks, as seawater medaka vitellogenin standard items are collected, wherein, 25mM Tris-HCl homogenate buffers are (interior
Aprotinin containing 0.5TIU/mL, pH 7.5.
The preparation method of described rabbit-anti seawater medaka vitellogenin antibody, comprises the following steps：
Take the seawater medaka vitellogenin of purifying, add isometric Freund's complete adjuvant, it is fully emulsified after be made
Immunoreagent, subcutaneous multi-point injection, every injection volume 0.1mL are carried out to male, healthy new zealand white rabbit；Then, every two
All booster immunization 1 time, single immunization dosage is 600 μ g, and using incomplete Freund's adjuvant it is fully emulsified after carry out subcutaneous multiple spot
Injection, continuous booster immunization 5 times；5 days Culling heart bloods after the 5th is immune, 6000r/min is centrifuged 20 minutes, collects blood plasma, is obtained
Rabbit-anti seawater medaka vitellogenin polyclonal antiserum；Isometric ice-cold saturated ammonium sulfate is added into antiserum molten
Liquid, 4 DEG C of concussions are centrifuged after 2 hours, and abandoning supernatant, precipitation 10mL PBSs dissolve；The solution of acquisition uses 0.45 μ
M membrane filtration, is subsequently added in affinity column, is eluted with PBS after 10 times of column volumes, with pH 2.7 0.1M
Glycine elution, that is, obtain rabbit-anti seawater medaka vitellogenin antibody.
The preparation method of the rabbit-anti seawater medaka vitellogenin antibody of described horseradish peroxidase-labeled, including
Following steps：
Weigh 6mg horseradish peroxidases to be dissolved in 2mL deionized waters, add the 0.1M's of the fresh configurations of 0.4mL
NaIO4Magnetic agitation 20 minutes under the conditions of solution, room temperature lucifuge；The solution of acquisition is fitted into bag filter, to 1mM pH 4.4
4 DEG C of lucifuges of sodium-acetate buffer dialyse 8-12 hours；Then, 0.5mL 0.16M glycol water, room are added thereto
Temperature is placed 30 minutes；3mg rabbit-anti seawater medaka vitellogenin antibody is added, and is well mixed, is fitted into bag filter, it is right
0.05M pH 9.5 4 DEG C of lucifuges of carbonate buffer solution are dialysed 8-12 hours；0.1mL 5mg/mL NaBH is added thereto4It is molten
Liquid, is placed 2 hours for 4 DEG C after being well mixed；It is slowly added to isometric saturated ammonium sulfate solution, abandoning supernatant after 4 DEG C of centrifugations,
Precipitation is dissolved using 0.15M pH 7.4 PBS；It is fitted into bag filter, the 8-12 that dialysed to 4 DEG C of lucifuges of PBS is small
When；3000r/min centrifugations obtain the rabbit-anti seawater medaka vitellogenin that supernatant is horseradish peroxidase-labeled and resisted
Body.
Marine environment estrogen pollution detection is carried out using mentioned reagent box, is comprised the following steps：
(1) rabbit-anti seawater medaka vitellogenin antibody is diluted to 5 μ g/mL using the coating buffer in box body, to 96 holes
The 100 μ L solution are added in each hole of ELISA Plate, 4 DEG C are coated with 8-12 hour, discard solution in hole, use cleaning solution washing 5 times；
(2) the μ L of confining liquid 300 are added to 96 hole elisa Plates, closes 1 hour at room temperature, discard solution in hole, use washing
Liquid is washed 5 times；
(3) seawater medaka vitellogenin standard items are diluted to 3.9 using sample diluting liquid, 7.8,15.62,
31.25th, 62.5,125 and 250ng/mL concentration gradient, by the amount in 100 μ L/ holes by the seawater medaka ovum of various concentrations gradient
Yellow fat phosphoprotein standard items and testing sample are separately added into the different hole of 96 hole elisa Plates, are incubated 1 hour at 37 DEG C, are discarded hole
Interior solution, is washed 5 times using cleaning solution；Described testing sample is seawater, is diluted within the concentration range of standard curve.
(4) sample diluting liquid in box body is used by the rabbit-anti seawater medaka yolk original egg of horseradish peroxidase-labeled
Bai Kangti dilutes 2500 times, adds and is incubated 1 hour at the dilution antibody 100 μ L, 37 DEG C per hole；
(5) 100 μ L nitrite ions are added per hole, 37 DEG C of lucifuges are reacted 10 minutes；
(6) reaction terminate after, per hole add 50 μ L terminate liquids, and with ELIASA determine 450nm wavelength under each hole extinction
Value, determining needs to complete in 15 minutes after termination of the reaction；
(7) calculate：Using the logarithm value of standard concentration as abscissa, light absorption value is that ordinate makees standard curve, calculates mark
The regression equation of directrix curve, substitutes into equation, calculating obtains testing sample concentration, multiplied by with dilute by the light absorption value of testing sample
Multiple is released, the actual concentrations of seawater medaka vitellogenin as in testing sample.
Beneficial effects of the present invention are as follows：
(1) this kit is detected using seawater medaka vitellogenin homologous antibody first, compared to other utilizations
The method that heterologous antibody is detected, has the advantages that sensitivity height, high specificity, experimental result reproduction degree are high.
(2) this kit detects seawater medaka vitellogenin using double antibodies sandwich standard measure, compared to other method tool
Have the advantages that operating method simplicity, quick, workload are small.
Brief description of the drawings
Fig. 1 is the testing result of kit of the invention to seawater medaka vitellogenin standard items.
Fig. 2 is kit of the invention to male and the quantitative testing result of female seawater medaka homogenate.
Embodiment
The present invention is done below into once illustrating.
A kind of kit quantitatively detected for seawater medaka vitellogenin, including 1 box body, box body built with：
96 1 piece of hole elisa Plates, coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 bottle of terminate liquid.It is characterized in that
The kit be also equipped with seawater medaka vitellogenin standard items 1, rabbit-anti seawater medaka vitellogenin antibody 1,
The rabbit-anti seawater medaka vitellogenin antibody 1 of horseradish peroxidase-labeled.
The kit is prepared to comprise the following steps：
(1) seawater medaka vitellogenin standard items are prepared；
(2) rabbit-anti seawater medaka vitellogenin antibody is prepared；
(3) the seawater medaka vitellogenin antibody of horseradish peroxidase-labeled is prepared；
Wherein, it is as follows that described (1) prepares seawater medaka vitellogenin standard items method：
The method induction seawater medaka synthesis vitellogenin of 17 beta estradiols is exposed using water body.By seawater medaka
Adult fish is placed in 5L glass aquarium, and 10 tail seawater medakas are put into each glass aquarium；The exposure of 17 beta estradiols
Concentration is 100 μ g/L, all changes water daily, and rejoins 17 beta estradiols of respective amount, to keep exposure concentrations constant；It is early
Evening feeding bait, keeps 25 DEG C of water temperature, natural lighting；Zebra fish is put into anaesthetize in 50% alcohol and put to death by exposure after 10 days,
Weigh, and add 3 times of volumes ice colds homogenate buffer (25mM Tris, include 0.07M NaCl and 0.5TIU/mL Aprotinins,
PH 7.6), ice bath homogenate, homogenate low-temperature centrifugation 10 minutes collects supernatant；The supernatant being collected into is used for gel filtration
Chromatograph (Sephacryl S-300), (0.07M NaCl and 0.5TIU/mL Aprotinins, pH are included with 25mM Tri-HCl buffer solutions
7.6) with 1mL/min flow velocity swash of wave chromatographic column, main eluting peak is collected；Eluent is added into DEAE-Sepharose anion again
Displacement chromatography post, (0.5TIU/mL is included with the 25mM Tris-HCl buffer solutions containing 0.07,0.1,0.2 and 1M NaCl respectively
Aprotinin, pH 7.5) continue to elute with 1mL/min flow velocity, each gradient washes 60min collects 0.2M eluting peaks, is sea
Water medaka vitellogenin standard items.
It is as follows that described (2) prepare rabbit-anti seawater medaka vitellogenin antibody method：
The seawater medaka vitellogenin for taking 800 μ g to purify, adds isometric Freund's complete adjuvant, it is fully emulsified after
Subcutaneous multi-point injection, every injection volume 0.1mL are carried out to male, healthy new zealand white rabbit；Then, every two weeks booster immunizations
1 time, single immunization dosage is 600 μ g, and using the fully emulsified rear subcutaneous multi-point injection of progress of incomplete Freund's adjuvant, it is co-continuous
Booster immunization 5 times；5 days Culling heart bloods after the 5th is immune, 6000r/min is centrifuged 20 minutes, collects blood plasma, obtains rabbit-anti seawater
Medaka vitellogenin polyclonal antiserum；Isometric ice-cold saturated ammonium sulfate solution, 4 DEG C of concussions are added into antiserum
Centrifuged after 2 hours, abandoning supernatant, precipitation 10mL PBSs dissolve；The solution of acquisition uses 0.45 μm of filter membrane mistake
Filter, is subsequently added in Hitrap Protein G affinity columns, is eluted with PBS after 10 times of column volumes, uses pH
2.7 0.1M glycine elutions, that is, obtain rabbit-anti seawater medaka vitellogenin antibody.
The seawater medaka vitellogenin antibody method that described (3) prepare horseradish peroxidase-labeled is as follows：
Weigh 6mg horseradish peroxidases to be dissolved in 2mL deionized waters, add the 0.1M's of the fresh configurations of 0.4mL
NaIO4Magnetic agitation 20 minutes under the conditions of solution, room temperature lucifuge；The solution of acquisition is fitted into bag filter, to 1mM pH 4.4
4 DEG C of dialysed overnights of sodium-acetate buffer；Then, 0.5mL 0.16M glycol water is added thereto, and room temperature places 30
Minute；The rabbit-anti seawater medaka vitellogenin antibody described in 3mg claims 1 is added, and is well mixed, loads bag filter
In, to 0.05M pH9.5 4 DEG C of dialysed overnights of carbonate buffer solution；0.1mL 5mg/mL NaBH is added thereto4Solution,
Placed 2 hours for 4 DEG C after well mixed；It is slowly added to isometric saturated ammonium sulfate solution, abandoning supernatant after 4 DEG C of centrifugations, profit
Precipitation is dissolved with 0.15M pH 7.4 PBS；It is fitted into bag filter, to 4 DEG C of dialysed overnights of PBS；3000r/
Min centrifugations obtain the rabbit-anti seawater medaka vitellogenin antibody that supernatant is horseradish peroxidase-labeled.
Finally, the seawater medaka vitellogenin standard items 1 of preparation, rabbit-anti seawater medaka vitellogenin are resisted
Body 1, the rabbit-anti seawater medaka vitellogenin antibody 1 of horseradish peroxidase-labeled, 1 piece of the hole enzyme mark version of blank 96,
And coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and terminate liquid each 1 it is bottled enter box body in, constitute the present invention
Kit.
Its concrete composition is as follows：
(1) 1 piece of the hole enzyme mark version of blank 96；
(2) seawater medaka vitellogenin standard items 1, required concentration is diluted to sample diluting liquid using preceding；
(3) rabbit-anti seawater medaka vitellogenin antibody 1,5 μ g/mL are diluted to coating buffer using preceding；
(4) the rabbit-anti seawater medaka vitellogenin antibody 1 of horseradish peroxidase-labeled, dilute using preceding use sample
Liquid is released by 1：2500 dilution proportion；
(5) coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 bottle of terminate liquid, described coating buffer is
The carbonate buffer solutions of 50mM pH 9.6：1.59g NaCO3And 2.93g NaHCO3It is dissolved in 1L distilled water；Cleaning solution be containing
0.05%Tween-20 150mM pH 7.4 phosphate buffer：8.0g NaCl,0.2g KCl,2.9g Na2HPO4·
12H2O,0.2g KH2PO4And 0.5mL Tween-20 are dissolved in 1L distilled water；Confining liquid is the phosphoric acid of the pH 7.4 containing 2%BSA
Salt buffer：0.2g BSA are dissolved in 10mL pH 7.4 phosphate buffer；Sample diluting liquid is containing 0.05%Tween-20
And 1%BSA phosphate buffer：0.1g BSA are dissolved in 10mL confining liquids；Nitrite ion is the limited public affairs of Nuo Bo Riders, Beijing science and technology
Take charge of the TMB one pack system nitrite ions of production；Terminate liquid is 2M H2SO4The aqueous solution.
This kit can be used for the monitoring of marine environment estrogen pollution, and following steps are divided into when using：
(1) rabbit-anti seawater medaka vitellogenin antibody is diluted to 5 μ g/mL using the coating buffer in box body, to 96 holes
The solution 100 μ L are added in each hole of plate, 4 DEG C of coatings are stayed overnight, and discard solution in hole, washed 5 times using cleaning solution；
(2) the μ L of confining liquid 300 are added into 96 orifice plates, is closed 1 hour at room temperature, is discarded solution in hole, use cleaning solution
Washing 5 times；
(3) seawater medaka vitellogenin standard items are diluted to 3.9 using sample diluting liquid, 7.8,15.62,
31.25th, 62.5,125 and 250ng/mL concentration gradient, adds standard concentration gradient into 96 orifice plates by 100 μ L/ holes or treats
Test sample product (suitably dilute), are incubated 1 hour at 37 DEG C, discard solution in hole, use cleaning solution to wash 5 times；
(4) sample diluting liquid 1 is used：The rabbit-anti seawater medaka yolk of 2500 times of dilution horseradish peroxidase-labeleds is former
Protein antibodies, add per hole and are incubated 1 hour at the dilution antibody 100 μ L, 37 DEG C；
(5) 100 μ L nitrite ions are added per hole, 37 DEG C of lucifuges are reacted 10 minutes；
(6) reaction terminate after, per hole add 50 μ L terminate liquids, and with ELIASA determine 450nm wavelength under each hole extinction
Value, determining needs to complete in 15 minutes after termination of the reaction；
(7) calculate：Using the logarithm value of standard concentration as abscissa, light absorption value is that ordinate makees standard curve, calculates mark
The regression equation of directrix curve, substitutes into equation by the light absorption value of sample, calculates sample concentration, multiplied by with extension rate, i.e.,
For the actual concentrations of seawater medaka vitellogenin in sample.
Claims (5)
1. a kind of kit quantitatively detected for seawater medaka vitellogenin, it is characterised in that described kit bag
1 box body is included, box body is built with 1 piece of hole elisa Plates of blank 96；1 seawater medaka vitellogenin standard items, is used using preceding
Sample diluting liquid is diluted to required concentration；1 rabbit-anti seawater medaka vitellogenin antibody, is diluted to using preceding with coating buffer
5μg/mL；The rabbit-anti seawater medaka vitellogenin antibody of 1 horseradish peroxidase-labeled, sample diluting liquid is used before use
By 1：2500 dilution proportion；Coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 bottle of terminate liquid；
Described coating buffer is carbonate buffer solution；Confining liquid, sample diluting liquid, cleaning solution are phosphate buffer；Nitrite ion
For one pack system nitrite ion；Terminate liquid is H2SO4The aqueous solution.
2. a kind of kit quantitatively detected for seawater medaka vitellogenin according to claim 1, its feature
It is, the carbonate buffer solution that described coating buffer is 50mM, pH 9.6；Described confining liquid is slow for the phosphate containing 2%BSA
Fliud flushing, pH 7.4；Described sample diluting liquid is the phosphate buffer containing 0.05%Tween-20 and 1%BSA；Described washes
Wash the phosphate buffer that liquid is the 150mM containing 0.05%Tween-20, pH 7.4；Described nitrite ion is aobvious for TMB one pack systems
Color liquid；Described terminate liquid is 2M H2SO4The aqueous solution.
3. the preparation method of the kit described in claim 1 or 2, it is characterised in that prepare the seawater medaka of tray interior
The rabbit-anti seawater of vitellogenin standard items, rabbit-anti seawater medaka vitellogenin antibody and horseradish peroxidase-labeled is blue or green
Medaka fish-egg xanthan protein antibodies, comprise the following steps：
The preparation method of described seawater medaka vitellogenin standard items, comprises the following steps：
1) the method induction seawater medaka synthesis vitellogenin of 17 beta estradiols is exposed using water body, wherein, 17 β-female two
The exposure concentrations of alcohol are 50~200 μ g/L；
2) the seawater medaka after exposure is taken, is weighed, the ice-cold homogenate buffer of seawater 3 times of quality of medaka is added, ice bath is even
Supernatant is collected after slurry, homogenate low-temperature centrifugation；Described homogenate buffer be 25mM Tris, include 0.07M NaCl and
0.5TIU/mL Aprotinins, pH 7.6；
3) by the supernatant gel permeation chromatography being collected into, with 25mM Tri-HCl homogenate buffer swash of wave elution chromatography posts,
Collect main eluting peak and obtain eluent, wherein, homogenate buffer includes 0.07M NaCl and 0.5TIU/mL Aprotinins, pH 7.6；
Eluent is added into anion exchange chromatography again, respectively with the 25mM Tris-HCl containing 0.07,0.1,0.2 and 1M NaCl
Homogenate buffer continues elution chromatography post, and each gradient washes 60min collects 0.2M eluting peaks, as seawater medaka yolk
Former protein standard substance, wherein, 0.5TIU/mL Aprotinins, pH 7.5 are included in 25mM Tris-HCl homogenate buffers；
The preparation method of described rabbit-anti seawater medaka vitellogenin antibody, comprises the following steps：
Take the seawater medaka vitellogenin of purifying, add isometric Freund's complete adjuvant, it is fully emulsified after be made it is immune
Reagent, subcutaneous multi-point injection, every injection volume 0.1mL are carried out to white rabbit；Every two weeks booster immunizations 1 time, single immunization dosage
For 600 μ g, and using incomplete Freund's adjuvant it is fully emulsified after carry out subcutaneous multi-point injection, continuous booster immunization 5 times；The 5th
5 days Culling heart bloods after immune, 6000r/min is centrifuged 20 minutes, collects blood plasma, obtains rabbit-anti seawater medaka vitellogenin many
Polyclonal antisera；Add isometric ice-cold saturated ammonium sulfate solution into antiserum, 4 DEG C of concussions are centrifuged after 2 hours, are discarded
Clear liquid, precipitation 10mL PBSs dissolve；It is added to after the solution filtering of acquisition in affinity column, with PBS
After elution, with pH 2.7 0.1M glycine elutions, that is, rabbit-anti seawater medaka vitellogenin antibody is obtained；
The preparation method of the rabbit-anti seawater medaka vitellogenin antibody of described horseradish peroxidase-labeled, including it is following
Step：
Weigh 6mg horseradish peroxidases to be dissolved in 2mL deionized waters, add the NaIO that 0.4mL concentration is 0.1M4Solution, room
After being stirred under the conditions of warm lucifuge, solution is fitted into bag filter, to 1mM pH 4.4 4 DEG C of lucifuges dialysis 8- of sodium-acetate buffer
12 hours；0.5mL 0.16M glycol water is added thereto, and room temperature adds 3mg rabbit-anti seawater medaka after placing
Vitellogenin antibody, and be well mixed, it is fitted into bag filter, 4 DEG C of lucifuges of carbonate buffer solution to 0.05M pH 9.5 are saturating
Analysis 8-12 hours；0.1mL 5mg/mL NaBH is added thereto4Solution, 4 DEG C of placements after being well mixed；It is slowly added to isometric
Saturated ammonium sulfate solution, 4 DEG C centrifugation after abandoning supernatant, utilize 0.15M pH 7.4 PBS dissolving precipitation；Load
In bag filter, 4 DEG C of lucifuges of PBS are dialysed 8-12 hours；It is horseradish peroxidating that 3000r/min centrifugations, which obtain supernatant,
The rabbit-anti seawater medaka vitellogenin antibody of thing enzyme mark.
4. the preparation method of kit according to claim 3, it is characterised in that seawater medaka vitellogenin standard
The preparation method step 3 of product) described in homogenate buffer swash of wave elution chromatography post flow velocity be 1mL/min.
5. the kit described in claim 1 or 2 is used to carry out marine environment estrogen pollution detection, it is characterised in that following step
Suddenly：
(1) rabbit-anti seawater medaka vitellogenin antibody is diluted to 5 μ g/mL using the coating buffer in box body, to 96 hole enzyme marks
The 100 μ L solution are added in each hole of plate, 4 DEG C are coated with 8-12 hour, discard solution in hole, cleaning solution is washed；
(2) the μ L of confining liquid 300 are added to 96 hole elisa Plates, closed 1 hour at room temperature, discard solution in hole, cleaning solution washing；
(3) seawater medaka vitellogenin standard items are diluted to 3.9 using sample diluting liquid, 7.8,15.62,31.25,
62.5th, 125 and 250ng/mL concentration gradient, by the amount in 100 μ L/ holes by the seawater medaka egg fat phosphorus of various concentrations gradient
Protein standard substance and testing sample are separately added into the different hole of 96 hole elisa Plates, are incubated 1 hour, are discarded molten in hole at 37 DEG C
Liquid, cleaning solution washing；Described testing sample is seawater, is diluted within the concentration range of standard curve；
(4) the rabbit-anti seawater medaka vitellogenin of horseradish peroxidase-labeled is resisted using the sample diluting liquid in box body
Body dilutes 2500 times, adds and is incubated 1 hour at the dilution antibody 100 μ L, 37 DEG C per hole；
(5) 100 μ L nitrite ions are added per hole, 37 DEG C of lucifuges are reacted 10 minutes；
(6) after reaction terminates, 50 μ L terminate liquids are added per hole, and the light absorption value in each hole under 450nm wavelength is determined with ELIASA, are surveyed
Need to complete in 15 minutes after termination of the reaction calmly；
(7) calculate：Using the logarithm value of standard concentration as abscissa, light absorption value is that ordinate makees standard curve, calculates standard bent
The regression equation of line, equation is substituted into by the light absorption value of testing sample, and calculating obtains testing sample concentration, multiplied by with dilution times
Number, the actual concentrations of seawater medaka vitellogenin as in testing sample.
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