CN107367617A - A kind of kit of the quantitative detection seawater medaka vitellogenin of improvement, preparation method and application - Google Patents

A kind of kit of the quantitative detection seawater medaka vitellogenin of improvement, preparation method and application Download PDF

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CN107367617A
CN107367617A CN201710519461.3A CN201710519461A CN107367617A CN 107367617 A CN107367617 A CN 107367617A CN 201710519461 A CN201710519461 A CN 201710519461A CN 107367617 A CN107367617 A CN 107367617A
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vitellogenin
medaka
seawater
seawater medaka
rabbit
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宫玉峰
易先亮
钟熙
张绍帅
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Dalian University of Technology
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

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Abstract

The present invention is a kind of kit of the quantitative detection seawater medaka vitellogenin of improvement, preparation method and application, belongs to environmental monitoring.Kit includes rabbit-anti seawater medaka vitellogenin antibody, coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 of the terminate liquid of 1 piece of hole elisa Plates of blank 96,1 seawater medaka lipovitellin standard items, 1 rabbit-anti seawater medaka vitellogenin antibody, 1 horseradish peroxidase-labeled.Seawater medaka lipovitellin has been isolated and purified by " gel permeation chromatography+ion-exchange chromatography " two-step method, the lipovitellin of acquisition has identical immunogenicity with vitellogenin, as the standard items of detection vitellogenin, and heat endurance is more preferably.This kit quickly, sensitively can quantitatively detect the vitellogenin in seawater medaka sample, and the biological monitoring that can be polluted for marine environment estrogen provides technological means.

Description

A kind of kit of the quantitative detection seawater medaka vitellogenin of improvement, prepare Method and application
Technical field
The invention belongs to environmental monitoring, is related to a kind of examination of the quantitative detection seawater medaka vitellogenin of improvement Agent box, there is more preferably detection repeatability and accuracy, the biological monitoring suitable for the pollution of marine environment estrogen.
Background technology
Environmental estrogens are the xenobiontics of a kind of wide variety, can upset the normal hormonal readiness of organism, damage Evil internal system function, and further influence reproductive process, population sex ratio and the scale of wild animal.In recent years, it is extra large Foreign environmental estrogens pollution problem causes increasing concern.
Vitellogenin (Vitellogenin, Vtg) is the precursor of oviparous animal livetin, in the thorn of 17 beta estradiols Under swashing, synthesized by liver with secreting, ovary is transported to by blood circulation, the nutrient source as embryonic development.Under normal circumstances, The raun that Vtg is generated the phase by yolk largely synthesizes, and milter can not synthesize or close because endogenous estrogen level is relatively low with juvenile fish It is very low into measuring.But milter is same with juvenile fish to have Vtg genes, in the presence of exogenous hormone, can also be induced to synthesize Vtg. Therefore, the Vtg levels of milter or juvenile fish are to indicate the ideal indicator of environmental estrogens pollution.
EUSA (Enzyme-linked immunosorbent assay, ELISA) is because with detection spirit The advantages of sensitivity is high, easy to operate, quick, have been widely used for Vtg detection.At present, Vtg ELISA detection methods have been established Fish be mostly fresh-water fishes, including zebra fish (Danio rerio), fathead minnow (Pimephales promelas), carp (Cyprinus carpio) and Gobiocypris rarus (Gobiocypris rarus) etc..Seawater medaka (Oryzias Melastigma) have it is small, be easy to raising, the generation cycle is short, salinity adaptation scope is wide, and react to environmental pollutants The characteristics of sensitive, it is preferable marine environmental monitoring model organism.
Research finds fish Vtg, particularly ocean fingerling Vtg, is also easily degraded even if preserving under cryogenic, And the Vtg protein fragments after degrading can increase its immunogenicity, so as to cause abnormal the becoming of ELISA experiment standard curves Change, influence the stability of ELISA testing results.And Vtg degradeds are also considered as the important original for causing ELISA accuracy relatively low Cause.Lipovitellin (Lipovitellin, Lv) is pyrolysis products of the Vtg in ovary, has similar immunogene to Vtg Property, and there is high heat endurance.Therefore, the antigen (standard items) in by the use of Lv as ELISA test experiences will significantly Increase the stability and accuracy of Vtg detections.However, it is at present that sea is established in antigen (standard items) exploitation there has been no research and utilization Lv Water medaka Vtg ELISA detection techniques, constrain application of the seawater medaka in marine environment estrogen pollution monitoring.
Therefore, purifying prepares the seawater medaka Lv of high-purity, establishes the seawater medaka Vtg using Lv as standard items ELISA quantitative detecting methods, the stability and accuracy of existing method are improved, there is important practical significance.
The content of the invention
The invention provides a kind of quantitative detection seawater medaka (Oryzias melastigma) the yolk original egg of improvement Existing detection can be greatly improved using seawater medaka lipovitellin as antigen standard in white kit, the kit The stability and accuracy of method.The detection range of this method is 7.8~1000ng/mL (R2>0.990), detection limit can reach To 3.1ng/mL, 3.85% and 4.83% are respectively smaller than with the coefficient of variation in group between group.
The technical scheme is that：
A kind of kit of quantitative detection seawater medaka (Oryzias melastigma) vitellogenin of improvement, should Kit includes 1 box body, and box body is built with 1 piece of hole elisa Plates of blank 96；1 seawater medaka lipovitellin standard Product, required concentration is diluted to sample diluting liquid using preceding；1 rabbit-anti seawater medaka vitellogenin antibody, uses preceding use Coating buffer is diluted to 5 μ g/mL；The rabbit-anti seawater medaka vitellogenin antibody of 1 horseradish peroxidase-labeled, before use 1 is pressed with sample diluting liquid：Dilution proportion；Coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and terminate liquid each 1 Branch.
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 mentioned reagent box, mainly prepare seawater medaka lipovitellin standard items in box body, Rabbit-anti seawater medaka vitellogenin antibody, the rabbit-anti seawater medaka vitellogenin of horseradish peroxidase-labeled resist Body, comprise the following steps：
The preparation method of described seawater medaka lipovitellin standard items, comprises the following steps：
1) take yolk to form the female seawater medaka ovary tissue in later stage, remove connective tissue, collect fish-egg；Add fish 3~4 times of volumes of ovum, the homogenate buffer of 4 DEG C of precoolings, after 4 DEG C of machines homogenate, 10000rpm/min is centrifuged 20 minutes, in collection Clear liquid.Described homogenate buffer is 25mM tromethamines (Tris), includes 0.07M NaCl and 0.5TIU/mL Aprotinins, And it is 7.6 to adjust pH value with HCl or NaOH.
2) (NH is slowly added into supernatant4)2SO4The saturation degree of powder to 70%, liquid of saltouing；After 10 hours, 4 At DEG C, centrifuged 10 minutes with 8000rpm/min, supernatant discarding, precipitation is redissolved in 25mM Tris-HCl, it is even to obtain ovum Starch extract solution；Contain 0.07M NaCl, pH 7.5 in described Tris-HCl.
3) 1mL ovum homogenate extract solution is taken to add to Sephacryl S-300 chromatographic columns, with the 25mM of the NaCl containing 0.07M Tris-HCl buffer solutions elute；Collect the eluting peak containing seawater medaka lipovitellin and carry out ion-exchange chromatography (DEAE-Sepharose Fast Flow), respectively with the 25mM containing 0.07M, 0.1M, 0.2M, 0.3M and 1.0M NaCl Tris-HCl buffer solutions are discontinuously eluted, and collect 0.2M elution fractions, as seawater medaka lipovitellin standard Product.Described Tris-HCl pH of buffer is 7.5.
Described rabbit-anti seawater medaka vitellogenin preparation method for antibody, 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.
Under conditions of natural lighting, 20~25 DEG C of water temperature, 17 beta estradiol exposure concentrations are 50~200 μ g/L, to sea Water medaka carries out exposing processing.
2) the seawater medaka after exposing 10 days is taken, is weighed, addition is the ice-cold homogenate buffering of 3 times of quality of seawater medaka 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) by the supernatant being collected into gel permeation chromatography (Sephacryl S-300), buffered with 25mM Tri-HCl Liquid collects main eluting peak with 1mL/min flow velocity swash of wave chromatographic column, wherein, 25mM Tri-HCl buffer solutions include 0.07M NaCl and 0.5TIU/mL Aprotinins, pH 7.6.Eluent is added into DEAE-Sepharose anion exchange chromatography again, used The 25mM Tris-HCl buffer solutions containing 0.07,0.1,0.2 and 1M NaCl continue to elute with 1mL/min flow velocity respectively, often Individual gradient washes 60min, 0.2M eluting peaks are collected, obtain seawater medaka vitellogenin, wherein 25mM Tris-HCl bufferings Liquid includes 0.5TIU/mL Aprotinins, pH 7.5.
4) the seawater medaka vitellogenin of purifying is taken, isometric Freund's complete adjuvant is added, is made after fully emulsified Into immunoreagent, 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, the dosage of single immunization reagent is 600 μ g, and uses the fully emulsified rear progress of incomplete Freund's adjuvant Subcutaneous multi-point injection, co-continuous booster immunization 5 times；5 days Culling heart bloods after the 5th is immune, 6000r/min are centrifuged 20 minutes, are received Collect blood plasma, obtain rabbit-anti seawater medaka vitellogenin polyclonal antiserum；Added into antiserum isometric ice-cold full And ammonium sulfate, 4 DEG C concussion 2 hours after centrifuge, abandoning supernatant, precipitation with 10mL PBSs dissolve；What is obtained is molten Liquid uses 0.45 μm of membrane filtration, is subsequently added in affinity column, after eluting 10 times of column volumes with PBS, uses PH 2.7 0.1M glycine elutions, 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 NaIO4Solution, magnetic agitation 20 minutes under the conditions of 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 dialysis 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 lucifuge dialysis 8-12 hours of carbonate buffer solution；0.1mL 5mg/mL NaBH is added thereto4It is molten Liquid, 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, it is small to 4 DEG C of lucifuge dialysis 8-12 of PBS 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) use the coating buffer in box body that rabbit-anti seawater medaka vitellogenin antibody is diluted into 5 μ g/mL, to 96 The 100 μ L solution are added in each hole of hole elisa Plates, 4 DEG C of coating 8-12 hours, discard solution in hole, use cleaning solution washing 5 It is secondary.
(2) the μ L of confining liquid 300 are added into 96 hole elisa Plates, closes 1 hour at room temperature, discards solution in hole, using washing Liquid is washed to wash 5 times.
(3) seawater medaka lipovitellin standard items are diluted to 3.9 using sample diluting liquid, 7.8,15.62, 31.25th, 62.5,125,250,500,1000 and 2000ng/mL concentration gradient, by the amount in 100 μ L/ holes by various concentrations gradient Seawater medaka lipovitellin standard items be separately added into testing sample in the different hole of 96 hole elisa Plates, incubated at 37 DEG C Educate 1 hour, discard solution in hole, washed 5 times using cleaning solution；Described testing sample is seawater, is diluted to standard curve Within concentration range.
(4) sample diluting liquid in box body is used by the rabbit-anti seawater medaka yolk original egg of horseradish peroxidase-labeled Bai Kangti dilutions times, the dilution antibody 100 μ L are added per hole, are incubated 1 hour at 37 DEG C.
(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 extinction in each hole under 450nm wavelength is determined with ELIASA Value, measure need 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, the light absorption value of testing sample is substituted into equation, calculates testing sample concentration, multiplied by with dilution times Number, the actual concentrations of seawater medaka vitellogenin as in testing sample.
Beneficial effects of the present invention are as follows：This kit passes through " gel permeation chromatography+ion-exchange chromatography " two-step method point From purifying seawater medaka lipovitellin, lipovitellin and the vitellogenin of acquisition have identical immunogene Property, the standard items using seawater medaka lipovitellin as detection vitellogenin, and heat endurance is more preferably.This kit The vitellogenin in seawater medaka sample quickly, sensitively can be quantitatively detected, can be that marine environment estrogen pollutes Biological monitoring provide technological means.Compared to existing method, the advantage of this kit is that there is more preferable testing result to reappear Property and accuracy.The detection range of this method is 7.8~1000ng/mL (R2>0.990), detection limit can reach 3.1ng/mL, Between group 3.85% and 4.83% are respectively smaller than with the coefficient of variation in group.
Brief description of the drawings
Fig. 1 is testing result of the kit to seawater medaka lipovitellin standard items of the present invention.
Embodiment
The present invention is done below into once illustrating.
A kind of kit of quantitative detection seawater medaka (Oryzias melastigma) vitellogenin of improvement, bag Include 1 box body, box body built with：(1) 96 1 piece of hole elisa Plates；(2) rabbit-anti seawater medaka vitellogenin antibody 1；(3) The rabbit-anti seawater medaka vitellogenin antibody 1 of horseradish peroxidase-labeled；(4) coating buffer, confining liquid, sample dilution Liquid, cleaning solution, nitrite ion and each 1 of terminate liquid.It is characterized in that the kit is also equipped with (5) seawater medaka egg fat phosphorus egg White standard items 1.
The kit is prepared to comprise the following steps：
(1) seawater medaka lipovitellin 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 lipovitellin standard items method：
Take yolk to form the female seawater medaka ovary tissue in later stage, remove connective tissue, collect fish-egg, add 3 times The homogenate buffer (25mM Tris, including 0.07M NaCl and 0.5TIU/mL Aprotinins, pH 7.6) of 4 DEG C of precoolings of volume is mixed Close, after 4 DEG C of machine homogenate, 10000g is centrifuged 20 minutes, collects supernatant；(NH is slowly added into supernatant4)2SO4Powder is extremely 70% saturation degree, liquid of saltouing；After 10 hours, at 4 DEG C, centrifuged 10 minutes, supernatant discarding, will precipitated again with 8000g 25mM Tris-HCl (including 0.07M NaCl, pH 7.5) are dissolved in, obtain ovum homogenate extract solution；1mL ovum are taken to be homogenized extract solution Add to Sephacryl S-300 chromatographic columns, eluted with the 25mM Tris-HCl (pH 7.5) of the NaCl containing 0.07M；Collection contains The eluting peak for having seawater medaka lipovitellin carries out ion-exchange chromatography (DEAE-Sepharose Fast Flow), point Do not carried out with the Tris-HCl buffer solutions (25mM, pH 7.5) containing 0.07M, 0.1M, 0.2M, 0.3M and 1.0M NaCl discontinuous Elution, collect 0.2M elution fractions, as seawater medaka lipovitellin standard items.
It is as follows that described (2) prepare rabbit-anti seawater medaka vitellogenin antibody 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, collect 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, 0.2M eluting peaks are collected, obtain sea Water medaka vitellogenin.
The seawater medaka vitellogenin for taking 800 μ g to purify, adds isometric Freund's complete adjuvant, after fully emulsified 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 are centrifuged 20 minutes, collect blood plasma, obtain 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, after eluting 10 times of column volumes with PBS, 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 NaIO4Solution, magnetic agitation 20 minutes under the conditions of 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 12 hours；Then, 0.5mL 0.16M glycol water, room temperature are added thereto Place 30 minutes；The rabbit-anti seawater medaka vitellogenin antibody described in 3mg claims 1 is added, and is well mixed, is loaded In bag filter, 0.05M pH 9.5 4 DEG C of lucifuges of carbonate buffer solution are dialysed 12 hours；0.1mL 5mg/mL are added thereto NaBH4Solution, placed 2 hours for 4 DEG C after being well mixed；Isometric saturated ammonium sulfate solution is slowly added to, is abandoned after 4 DEG C of centrifugations Supernatant is removed, dissolves precipitation using 0.15M pH 7.4 PBS；It is fitted into bag filter, to 4 DEG C of lucifuges of PBS Dialysis 12 hours；3000r/min centrifugations obtain the rabbit-anti seawater medaka yolk that supernatant is horseradish peroxidase-labeled Former protein antibodies.
Finally, by the seawater medaka lipovitellin standard items 1 of preparation, rabbit-anti seawater medaka vitellogenin Antibody 1, the rabbit-anti seawater medaka vitellogenin antibody 1 of horseradish peroxidase-labeled, the hole enzyme mark version 1 of blank 96 Block, and coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and terminate liquid each 1 it is bottled enter box body in, form this hair Bright kit.
Its concrete composition is as follows：
(1) 1 piece of the hole enzyme mark version of blank 96；
(2) seawater medaka lipovitellin 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, using preceding dilute with sample Liquid is released by 1：4000 dilution proportion；
(5) coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 bottle of terminate liquid, described coating buffer are 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, be divided into following steps during use：
(1) coating buffer in box body is used to dilute rabbit-anti seawater medaka vitellogenin antibody to 5 μ g/mL, to 96 holes The solution 100 μ L are added in each hole of plate, 4 DEG C of coatings overnight, are discarded 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 lipovitellin standard items are diluted to 3.9 using sample diluting liquid, 7.8,15.62, 31.25th, 62.5,125,250,500,1000 and 2000ng/mL concentration gradient, 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 using cleaning solution；Described testing sample is seawater, is diluted within the concentration range of standard curve.
(4) sample diluting liquid 1 is used：The rabbit-anti seawater medaka yolk of 4000 times of dilution horseradish peroxidase-labeleds is former Protein antibodies, the dilution antibody 100 μ L are added per hole, are incubated 1 hour at 37 DEG C；
(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 extinction in each hole under 450nm wavelength is determined with ELIASA Value, measure need 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, the light absorption value of sample is substituted into equation, calculates sample concentration, multiplied by with extension rate, i.e., For the actual concentrations of seawater medaka vitellogenin in sample.The actual concentrations of this vitellogenin can reflect female in seawater The pollution situation of hormone, is widely used in environmental monitoring.

Claims (6)

  1. A kind of 1. kit of the quantitative detection seawater medaka vitellogenin of improvement, it is characterised in that described kit Including 1 box body, box body is built with 1 piece of hole elisa Plates of blank 96；1 seawater medaka lipovitellin standard items, use It is preceding to be diluted to required concentration with sample diluting liquid；1 rabbit-anti seawater medaka vitellogenin antibody, using preceding dilute with coating buffer Release to 5 μ g/mL；The rabbit-anti seawater medaka vitellogenin antibody of 1 horseradish peroxidase-labeled, using preceding dilute with sample Liquid is released by 1：2500 dilution proportion；Coating buffer, confining liquid, sample diluting liquid, cleaning solution, nitrite ion and each 1 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. 2. a kind of kit of the quantitative detection seawater medaka vitellogenin of improvement according to claim 1, it is special Sign is, described coating buffer is 50mM carbonate buffer solution, pH 9.6；Described confining liquid is the phosphate containing 2%BSA Buffer solution, pH 7.4；Described sample diluting liquid is the phosphate buffer containing 0.05%Tween-20 and 1%BSA；Described Cleaning solution be the 150mM containing 0.05%Tween-20 phosphate buffer, pH 7.4；Described nitrite ion is TMB one pack systems Nitrite ion；Described terminate liquid is 2M H2SO4The aqueous solution.
  3. 3. the preparation method of the kit described in claim 1 or 2, it is characterised in that prepare the seawater medaka yolk in box body Fat phosphoprotein standard items, rabbit-anti seawater medaka vitellogenin antibody, the rabbit-anti seawater green grass or young crops Medaka of horseradish peroxidase-labeled Fish-egg xanthan protein antibodies, comprise the following steps：
    The preparation method of described seawater medaka lipovitellin standard items, comprises the following steps：
    1) take yolk to form the female seawater medaka ovary tissue in later stage, remove connective tissue, collect fish-egg；Add fish-egg 3 ~4 times of volumes, the homogenate buffer of 4 DEG C of precoolings, after 4 DEG C of machines homogenate, 10000rpm/min is centrifuged 20 minutes, collects supernatant Liquid；Described homogenate buffer is 25mM tromethamine Tris, includes 0.07M NaCl and 0.5TIU/mL Aprotinins, is used in combination HCl or NaOH adjustment pH value is 7.6；
    2) (NH is slowly added into supernatant4)2SO4The saturation degree of powder to 70%, liquid of saltouing；After 10 hours, at 4 DEG C, Centrifuged 10 minutes, abandoning supernatant, precipitation is redissolved in 25mM Tris-HC buffer solutions with 8000rpm/min, obtained Ovum is homogenized extract solution；
    3) take 1mL ovum homogenate extract solution to add to chromatographic column, eluted with the 25mM Tris-HCl buffer solutions of the NaCl containing 0.07M； Collect eluting peak containing seawater medaka lipovitellin and carry out ion-exchange chromatography, respectively with containing 0.07M, 0.1M, 0.2M, 0.3M and 1.0M NaCl 25mM Tris-HCl buffer solutions are discontinuously eluted, and collect 0.2M elution fractions, are Seawater medaka lipovitellin standard items；
    The preparation method of described rabbit-anti seawater medaka vitellogenin antibody, 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, and adds the homogenate buffer of 3 times of quality of seawater medaka, ice bath homogenate, Supernatant is collected after homogenate low-temperature centrifugation；
    3) the supernatant gel permeation chromatography that will be collected into, with 25mM Tri-HCl homogenate buffer swash of wave elution chromatography posts, Collect main eluting peak and obtain eluent, wherein, 25mM Tri-HCl homogenate buffers include 0.07M NaCl and 0.5TIU/mL suppression Peptase, pH 7.6；Eluent is added into anion exchange chromatography again, respectively with containing 0.07,0.1,0.2 and 1M NaCl's 25mM Tris-HCl homogenate buffers continue swash of wave elution chromatography post, each gradient washes 60min, collect 0.2M eluting peaks, obtain Seawater medaka vitellogenin is obtained, wherein, 25mM Tris-HCl homogenate buffers include 0.5TIU/mL Aprotinins, pH 7.5；
    4) seawater medaka vitellogenin is taken, isometric Freund's complete adjuvant is added, immunoreagent is made after fully emulsified, Subcutaneous multi-point injection, every injection volume 0.1mL are carried out to white rabbit；Every two weeks booster immunizations 1 time, the dosage of single immunization reagent 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 are centrifuged 20 minutes, collect blood plasma, and it is more to obtain rabbit-anti seawater medaka vitellogenin Polyclonal antisera；Add isometric saturated ammonium sulfate solution into antiserum, 4 DEG C of concussions centrifuge after 2 hours, supernatant discarding Liquid, precipitation 10mL PBSs dissolve；Add in affinity column after the solution filtering of acquisition, eluted with PBS Afterwards, with pH 2.7 0.1M glycine elutions, 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 temperature, lucifuge, solution is fitted into bag filter, with 1mM pH4.4 4 DEG C of lucifuge 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 ovum after placing Xanthan protein antibodies, and be well mixed, it is fitted into bag filter, is dialysed with 0.05M pH 9.5 4 DEG C of lucifuges of carbonate buffer solution 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, abandoning supernatant after 4 DEG C of centrifugations, dissolve precipitation using 0.15M pH 7.4 PBS；Load saturating Analyse in bag, with 4 DEG C of lucifuge dialysis 8-12 hours of PBS；It is horseradish peroxidase that 3000r/min centrifugations, which obtain supernatant, The rabbit-anti seawater medaka vitellogenin antibody of enzyme mark.
  4. 4. the preparation method of kit according to claim 3, it is characterised in that seawater medaka lipovitellin mark Contain 0.07MNaCl, pH 7.5 in step 2) and step 3) Tris-HCl buffer solutions described in the preparation method of quasi- product.
  5. 5. the preparation method of the kit according to claim 3 or 4, it is characterised in that rabbit-anti seawater medaka yolk is former The flow velocity of step 3) homogenate buffer swash of wave elution chromatography post described in the preparation method of protein antibodies is 1mL/min.
  6. 6. 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) use the coating buffer in box body that rabbit-anti seawater medaka vitellogenin antibody is diluted into 5 μ g/mL, to 96 hole enzymes The 100 μ L solution are added in each hole of target, 4 DEG C of coating 8-12 hours, solution in hole is discarded, is washed using cleaning solution；
    (2) the μ L of confining liquid 300 are added into 96 hole elisa Plates, closes 1 hour at room temperature, solution in hole is discarded, using cleaning solution Washing；
    (3) seawater medaka lipovitellin standard items are diluted to 3.9 using sample diluting liquid, 7.8,15.62,31.25, 62.5th, 125,250,500,1000 and 2000ng/mL concentration gradient, by the amount in 100 μ L/ holes by the seawater of various concentrations gradient Medaka lipovitellin standard items and testing sample are separately added into the different hole of 96 hole elisa Plates, and it is small that 1 is incubated at 37 DEG C When, solution in hole is discarded, is washed using cleaning solution；Described testing sample is seawater, is diluted to the concentration range of standard curve Within；
    (4) sample diluting liquid in box body is used to resist the rabbit-anti seawater medaka vitellogenin of horseradish peroxidase-labeled Body dilutes 2500 times, and the dilution antibody 100 μ L are added per hole, are incubated 1 hour at 37 DEG C；
    (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, and it is bent to calculate standard The regression equation of line, the light absorption value of testing sample is substituted into equation, calculates testing sample concentration, multiplied by with extension rate, The actual concentrations of seawater medaka vitellogenin as in testing sample.
CN201710519461.3A 2017-06-30 2017-06-30 A kind of kit of the quantitative detection seawater medaka vitellogenin of improvement, preparation method and application Pending CN107367617A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN109254156A (en) * 2018-10-23 2019-01-22 山东出入境检验检疫局检验检疫技术中心 Detect immune-blotting method kit and its application of Pseudopleuronectes fish vitellogenin

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JP2001122899A (en) * 1998-11-10 2001-05-08 Sumitomo Chem Co Ltd Vitellogenin originating from cyprinodontiformes and antibody
CN101398425A (en) * 2008-10-23 2009-04-01 中国海洋大学 Kit for detecting ocean incretion harass chemical substance, preparation method and application
CN103033631A (en) * 2012-12-20 2013-04-10 中国海洋大学 Test kit for qualitatively detecting fish vitellogenin by utilizing lipovitellin antibody
CN103184276A (en) * 2011-12-30 2013-07-03 中国科学院城市环境研究所 Water estrogen pollution detection method based on oryzias melastigma vitellogenin
CN104345156A (en) * 2014-11-14 2015-02-11 中国水产科学研究院黄海水产研究所 Fat greenling vitellogenin sandwich ELISA kit and preparation method, detection method and application thereof
CN104597247A (en) * 2014-12-09 2015-05-06 中国海洋大学 Danio reeio vitellogenin sandwich ELISA kit, detection method and application thereof

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Publication number Priority date Publication date Assignee Title
JP2001122899A (en) * 1998-11-10 2001-05-08 Sumitomo Chem Co Ltd Vitellogenin originating from cyprinodontiformes and antibody
CN101398425A (en) * 2008-10-23 2009-04-01 中国海洋大学 Kit for detecting ocean incretion harass chemical substance, preparation method and application
CN103184276A (en) * 2011-12-30 2013-07-03 中国科学院城市环境研究所 Water estrogen pollution detection method based on oryzias melastigma vitellogenin
CN103033631A (en) * 2012-12-20 2013-04-10 中国海洋大学 Test kit for qualitatively detecting fish vitellogenin by utilizing lipovitellin antibody
CN104345156A (en) * 2014-11-14 2015-02-11 中国水产科学研究院黄海水产研究所 Fat greenling vitellogenin sandwich ELISA kit and preparation method, detection method and application thereof
CN104597247A (en) * 2014-12-09 2015-05-06 中国海洋大学 Danio reeio vitellogenin sandwich ELISA kit, detection method and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109254156A (en) * 2018-10-23 2019-01-22 山东出入境检验检疫局检验检疫技术中心 Detect immune-blotting method kit and its application of Pseudopleuronectes fish vitellogenin

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