CN1285912C - Kit for detecting environmente female hormone effect and its preparation method - Google Patents
Kit for detecting environmente female hormone effect and its preparation method Download PDFInfo
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- CN1285912C CN1285912C CN 200310115782 CN200310115782A CN1285912C CN 1285912 C CN1285912 C CN 1285912C CN 200310115782 CN200310115782 CN 200310115782 CN 200310115782 A CN200310115782 A CN 200310115782A CN 1285912 C CN1285912 C CN 1285912C
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Abstract
The present invention discloses a reagent box for detecting the effect of environmental estrogen and a preparation method thereof. The reagent box for detecting the effect of environmental estrogen of the present invention comprises a polyclonal antibody of crucian vitellogenin and a monoclonal antibody of crucian vitellogenin, wherein the polyclonal antibody of crucian vitellogenin is covered on an enzyme target plate, and the monoclonal antibody of crucian vitellogenin is marked with enzymes. The preparation method of the reagent box comprises the following steps: 1) the polyclonal antibody of crucian vitellogenin is prepared, is covered on the enzyme target plate, and is sealed by BSA; 2) the monoclonal antibody of crucian vitellogenin is prepared and is marked with enzymes, and the marked antibody is mixed with same amounts of glycerol; 3) the polyclonal antibody of crucian vitellogenin, which is obtained in step 1), and the monoclonal antibody of crucian vitellogenin, which is obtained in step 2), are jointly packed so as to obtain the reagent box for detecting the effect of environmental estrogen. The reagent box of the present invention provides a significant analysis means and a tool for detecting possible pollution behavior of the environmental estrogen in water environment.
Description
Technical field
The present invention relates to kit of a kind of testing environment estrogen effect and preparation method thereof.
Background technology
Along with the develop rapidly of industrial or agricultural, a large amount of inorganicss, organism constantly are produced, and environment carries the waste material of extraneous discharging at any time as the basis of human and various biological existences.The sixties in 20th century, human these toxic and harmful substances that just come to realise are to the human and biological various adverse effects that produced, especially to the influence of aquatic ecological environment, because the final home to return to of most of chemical substance is a water environment, approach is directly discharging or enters water environment by the mutual exchange process of aqueous vapor.Chemical pollutant not only destroys the value of natural resources, for example DDTs can endanger existence and the procreation of various aquatic birds and fish by food chain, especially to those high trophic level kinds, even make that by atmospheric air circulation arctic regions biology (as polar bear) is subjected to threaten equally, this finally can cause the collapse of the ecosystem, harm humans itself.
In the middle of these chemical substances, those can disturb the normal endocrine material of biosome to be called as incretion interferent (EDCs) with different approaches, as cause the chemical substance of estrogen effect, antiestrogenic effect, androgen effect, antiandrogen effect etc.The ambient level of these materials is very low, and the damaging effect of ecology is had only by long term exposure and can be observed, and therefore the mankind's harm is difficult in a short time and is judged.In case and damaging effect becomes obviously, consequence also just is difficult to control so.This just requires to set up a kind of early stage warning system (biomarker) and is used as the signal that the ecosystem that environmental pollution causes is about to wreck.When environment changes, when especially being subjected to the external source pollution, high-caliber biochemical the variation can change prior to early stage biology, and biomarker just can be from molecule, cell and organized the variation of the physiological parameter that reflects whole biosome on the level in time, exactly, biochemical composition, cytogenetics material.Because the biomarker between the different tissues can be used as noxious material and enters organic early warning signal, when this micro-variations is accumulated to a certain degree, can cause the variation of body, thereby make that setting up a kind of early stage biomarker reflects that in time biosome may be varied to for possibility by imminent biology.Toxicity test has the limitation of self for environmental organism monitoring (BM), this be because they can not the reasonable dismissal chemical substance toxicological effect after distribution behavior, metabolism behavior, absorption behavior, accumulation behavior in environment and and the metabolism itself.A kind of special contact of setting up between biomarker reaction and the living organism feature (as survival, breeding) will provide one to have far-reaching basis in the application aspect the environmental evaluation for biomarker.
For how selecting monitoring bio, should select those widely distributed species according to analysis purpose, the consideration of some others also will be arranged simultaneously.In water environment, fish is transferred to high trophic level biology with energy from low trophic level biology as the transmitter of energy, can be caught widely simultaneously.Fish to noxious material in the correct understanding environment in vivo absorption, metabolism behavior and the biochemical reactions that causes significance is arranged, therefore, fish are chosen as the environmental monitoring biology.Most of biomarkers seem to be applied directly to a certain specific fish health check-up and survey, but consider that physiologic characters different between the different fish and environmental pollution cause different biomarker reactions, so need to select multiple class to detect, thereby provide reliability data for correctly estimating ecologic environment with additional mutually.
In theory, a kind of pollutant stress cause the one-level biologically under the normal condition, also just can be used as a kind of biomarker.And exceeding one when delimiting (pollutant dosage and exposure duration) when pollutant level, pollutant-dose response will break away from normal range, makes biosome demonstrate the multiple action effect on higher level.This just need be to biological the early stage early warning system of contaminated foundation.And biomarker can be regarded as the early stage physical biochemical reaction that biosome is subjected to certain toxicity material exposure back generation, and this can be used as the indicator of biological respinse theoretically, for having built bridge between exterior materials exposure and the body internal reaction.In general, the biomarker on the cellular level has more universal significance with respect to biological organization, because for biosome, various biochemical reactions are closely similar.Therefore good biomarker should be able to reflect the delicate variation that biosome is taken place sensitive, fast.In health risk assessment, after biosome was subjected to the effect of environment xenobiotic pollutants, biomarker just can be used to illustrate cause-effect relationship between them, dose-effect relationship etc.It is generally acknowledged that biomarker is that the reflection biosome is subjected to the middle compensatory reaction of physiological effect that external source is polluted and taken place.After these compensatory reactions began to take place, the possibility of organism survival just began to descend.Because when biosome ran into the living environment change, the compensatory reaction that begins to start just began to have threatened the safety of biosome self.Utilize biomarker carry out the environmental monitoring biggest advantage be exactly they can for pollutant the follow-up biology that may cause change useful information be provided, and be not only to quantize the environmental contaminants levels.
Under the normal condition, oviparity female body inner estrogen level raises before mating period, cell cultured supernatant secretion Vtg (Vitellogenin, VTG), VTG enters ovary by blood circulation, in absorption, the transhipment of cystencyte after filtration, is taken in the archiblast by the Microvillares micro-pinocytosis of egg mother cell itself at last, be decomposed to form vitellin and phosvitin, with source as the early stage of embryonic development endogenous nutrition and energy.This process mainly occurs in the later stage that egg mother cell is grown, and the volume of egg mother cell sharply increases during the grand period of growth, the accumulation of yolk is consistent with sexual gland.Wherein estrogen plays an important regulatory role, promptly in the formation of the generation of sexual gland, growth, gamete, maturation, play excite, the endocrine effect of feedback regulation.Can not take place in this process male creature body, but still have estrogen receptor and VTG expressing gene in the male creature liver cell nuclear.When having estrogen and class estrogen substance in the environment, will be with same pathway stimulation male creature liver cell secretion VTG, owing to lack normal metabolism, decomposition approach, the unusual VTG that produces is (especially male creature) accumulation in vivo, hinders normal metabolism.Simultaneously, female/class estrogen substance also can produce interference effect to the normal internal system of biosome, causes a series of physiology pathology, and threatens the procreation of biotic population and health, the sustainable development of ecologic environment.And every kind of fish VTG molecular weight, structure sex character are different (confirming by SDS-PAGE and Western blotting method), and this VTG that has illustrated that the fish body is produced has kind of the specificity of (or genus).So just need prepare different specific antibodies, to be used for reflecting that accurately there is level in environmental estrogens at fish not of the same race.Crucian carp be a kind ofly distribute extensively, easily catch, the fish of ubiquity.
Immune system is as the main defence organization at the intrusion material, and all allogenic materials (being called antigen) all can stimulate body immune system to produce responsing reaction targetedly, produce specific antibody.The specificity of antigen and antibody is in conjunction with impelling body to produce series reaction, and it is external to make that finally antigenic substance is gone out by organism metabolism, and body recovery is normal.Many molecules can both cause immune response, and every kind of antigen molecule has its unique shape, thereby cause the antigen and antibody specific reaction.Less antigen may have only an epitope, and the molecule that basis big, the complicated presumable antibody-AI of molecule is two complementary shape just in time can match.The ELISA method is exactly according to this antigen--the specificity combination between antibody, enzyme labeling on the antibody/antigen molecule, is formed enzyme labelled antibody/enzyme-labelled antigen, and be enzyme conjugates.This enzyme conjugates issues in the catalytic action of enzyme that unboiled water is separated, oxidation reaction, generate coloured product,, and reflect the amount of tested antigen because the activity of enzyme conjugates is directly proportional with the depth that product presents, therefore according to the having or not and the depth of color, locate or quantitative antigen.And, just can judge the ecology influence that has level and may bring of environmental hormone in the water environment according to the generation antigen levels.
The innovation and creation content
The kit that the purpose of this invention is to provide a kind of estrogen effect of testing environment more delicately.
The kit of testing environment estrogen effect provided by the present invention, it comprises the crucian carp Vtg polyclonal antibody that is coated on the ELISA Plate, two of crucian carp Vtg monoclonal antibody and enzyme labeling resists.
For more convenient execute-in-place, described kit also comprises crucian carp Vtg, oxygenant, developer and the quality controlled serum of normal concentration.
Described crucian carp Vtg polyclonal antibody is crucian carp Vtg rabbit polyclonal antibody preferably; Described crucian carp Vtg monoclonal antibody is crucian carp Vtg mouse resource monoclonal antibody preferably; The sheep anti mouse two of two anti-preferably horseradish peroxidase-labeled of described enzyme labeling is anti-.
Described crucian carp Vtg polyclonal antibody, crucian carp Vtg monoclonal antibody and two resist and all can prepare according to a conventional method.
Described ELISA Plate can be 96 hole wall scroll ELISA Plate, also can be two removable ELISA Plate of bar.
Second purpose of the present invention provides a kind of method for preparing the kit of above-mentioned testing environment estrogen effect.
The method of the kit of the above-mentioned testing environment estrogen effect of preparation provided by the present invention may further comprise the steps:
1) preparation crucian carp Vtg polyclonal antibody, and bag is sealed with BSA by on ELISA Plate;
2) prepare crucian carp Vtg monoclonal antibody, and mix with equivalent glycerine;
3) preparation resists with two of enzyme labeling;
4) with the crucian carp Vtg polyclonal antibody, the step 2 that obtain in the step 1)) in the crucian carp Vtg monoclonal antibody that obtains and two of the usefulness enzyme labeling that obtains in the step 3) anti-ly packed jointly, obtain the kit of testing environment estrogen effect.
Two of described crucian carp Vtg polyclonal antibody, crucian carp Vtg monoclonal antibody and enzyme labeling resists and all can prepare according to a conventional method.Described crucian carp Vtg polyclonal antibody is crucian carp Vtg rabbit polyclonal antibody preferably; Described crucian carp Vtg monoclonal antibody is crucian carp Vtg mouse resource monoclonal antibody preferably; The sheep anti mouse two of two anti-preferably horseradish peroxidase-labeled of described enzyme labeling is anti-.
The present invention utilizes crucian carp Vtg and binding immunoassay to learn the ELISA reaction mechanism dexterously, provide a kind of easy, efficiently, the kit of estrogen effect in the testing environment delicately.For other biological species, crucian carp distributes very extensive in China, and adaptive faculty is extremely strong, almost distribution is all arranged in each waters, therefore draw materials for the investigation of environmental estrogens great convenience is provided, also for judging that different water environments are subjected to the environmental estrogens pollution and provide indispensable biomaterial to the influence that biology causes.The kit of estrogen effect is easy and simple to handle, quick in the testing environment of the present invention, does not have special instrument requirement, and the sample requirement is few, thereby greatly reduces analysis cost and improved analysis efficiency.Kit of the present invention provides important analysis means for detecting the environmental estrogens pollution behavior that may exist in China's water environment, also provides a kind of new instrument for analyzing the influence that healthy suffered influence of biosome in the water environment and environmental estrogens may cause ecology.The present invention is with a wide range of applications, and especially is with a wide range of applications for those laboratory and scientific research institutions that do not possess large-sized analytic instrument.
The present invention will be further described below in conjunction with drawings and the specific embodiments.
Description of drawings
Fig. 1 is that kit of the present invention detects the usefulness curve
Embodiment
Embodiment 1, crucian carp Vtg MONOCLONAL ANTIBODIES SPECIFIC FOR
The crucian carp Vtg is expelled to BalB/C mouse limb week, back multiple spot after emulsification (Freund's complete adjuvant and Freund), dosage is 50 μ g/0.5ml/, and per 10 days once, altogether immunity four times (only exempting from 25 μ g/0.5ml/ for spleen for the last time); Cultivate simultaneously myeloma cell Sp2/0 to cell be satisfactory, bright, individual evenly after, get the immune mouse spleen, merge 37 ℃, HAT nutrient culture media, CO with myeloma cell Sp2/0
2Incubator is cultivated, and changes half liquid in per 2 days, and indirect ELISA screens positive hybridoma cell and changes the HT nutrient solution after 2 weeks.To screen gained positive cell clone cultivation (indirect ELISA detection), the frozen part cell in back is cultivated in extension, and part is injected in the mouse body, to induce the ascites that contains antibody.Get ascites after 10 days continuously, centrifugal: 4500rpm/min, 30min, ascites supernatant are stored in-80 ℃ of ultra low temperature freezers.Purification ascites adopts sad-saturated ammonium sulfate method, and purification antibody is dialysed more than 24 hours under PBS, 4 ℃ of conditions, and liquid is changed 3-4 time in the centre.Use BaCl
2Detect and do not have the precipitation generation.With antibody and equivalent glycerine mixing, packing is kept in-20 ℃ of refrigerators then.The ELISA method is measured the affinity constant K of antigen-antibody
AffBe 7.0 * 10
8L/mol.
The preparation of embodiment 2, crucian carp Vtg ELISA kit
The crucian carp Vtg rabbit polyclonal antibody bag that at first will prepare according to a conventional method is by on the removable ELISA Plate of wall scroll (polystyrene board), the BSA sealing, behind 37 ℃, 2h, washing, place 4 ℃ of condition lower seals preserve, standby.The crucian carp Vtg normal concentration of prepare laboratory-induced simultaneously, purifying and preparing.With above-mentioned antigen, crucian carp Vtg rabbit polyclonal antibody, crucian carp Vtg mouse resource monoclonal antibody, HRP mark sheep anti mouse are two anti-, chromogenic substrate (TMB), stop buffer, cleansing solution (Tween20 of PBS+0.05% volume), crucian carp negative serum are packaged to be kit, and every kind of reagent is respectively in the kit: the ELISA Plate of crucian carp Vtg polyclonal antibody bag quilt; Crucian carp Vtg mouse resource monoclonal antibody 100 μ l (diluting 1000 times of uses); Crucian carp Vtg normal concentration 100 μ l (1mg/ml); HRP mark two anti-(diluting 2500 times of uses); Colour developing liquid: PB, substrate (TMB), hydrogen peroxide (1000: 10: 1.5 times of uses during use); Stop buffer (2mol/L H
2SO
4) be 5ml; PBS (being made into 2L uses); TWEEN20:1.0ml; The crucian carp negative serum is 100 μ l (diluting 500 times of uses).
Embodiment 3, crucian carp Vtg ELISA kit are to the detection of environmental estrogens effect level of pollution
The kit that obtains with embodiment 2 induces carp, sturgeon and the wolffish that catches from environment, mullet, perch, copper fish to intersect mensuration to E2, the Vtg that may have in the analytic sample.Detailed process is as follows:
Select E2 to induce carp, crucian carp, sturgeon and the wolffish that from environment, catches, mullet, perch, copper fish, blood drawing separate serum, cryopreservation is equipped with fixing preservation of organization material in biochemical analysis simultaneously.During working sample, the sample that the crucian carp Vtg of normal concentration is diluted to 0.5,0.8,1.0,1.2,1.4 μ g/ml and suitably dilution adds (100 μ l/ hole) in the ELISA Plate simultaneously, 37 ℃ are washed after hatching 1h, add crucian carp Vtg mouse resource monoclonal antibody and (be diluted to 1000 times, 100 μ l/ holes), hatch 1h for 37 ℃, the back washing, two anti-(2500 times of the 2-3% skimmed milk power dilutions that add horseradish peroxidase-labeled, 100 μ l/ holes), hatch 1h for 37 ℃, washing, add substrate solution (100 μ l/ hole) dark place reaction 10-15min, add 2M H
2SO
4Stop buffer (50 μ l/ hole), microplate reader (450nm) colorimetric estimation absorbance (OD
450nm), according to Vtg content: OD in the quantitative sample of typical curve
(sample)=OD
(sample)-2OD
(feminine gender)The result shows the intersection obvious (66.9%) of carp, and sturgeon and wolffish, mullet, perch, copper fish then do not have significantly intersection (using HPLC to detect the VTG with certain level).This illustrates that this kit has very strong specificity.
By this kit, the Vtg that may contain in the detection crucian carp serum (or blood plasma) that can be quick, sensitive only needed just can obtain a result in 3-4 hour, and sensitivity can reach the 50ng/ hole.It detects usefulness as shown in Figure 1, and the result shows the range of linearity of this method at 500-1400ng/ml, and the recovery is at 95.95-108.5%.Needed sample size during mensuration (serum or blood plasma) is considerably less, gets final product about about 100 μ l.VTG is two sandwich method for determining crucian carp Vtg typical curves among Fig. 1: Y=0.7175X-0.2971 (R
2=0.98), prepares normal concentration antigen (0.5,0.8,1.0,1.2,1.4 μ g/ml).
Claims (10)
1, a kind of kit of testing environment estrogen effect, it comprises the crucian carp Vtg polyclonal antibody that is coated on the ELISA Plate, crucian carp Vtg monoclonal antibody, two of enzyme labeling resists oxygenant and developer.
2, kit according to claim 1 is characterized in that: described enzyme labeling is a horseradish peroxidase-labeled.
3, kit according to claim 1 and 2 is characterized in that: described kit also comprises the crucian carp Vtg of normal concentration.
4, kit according to claim 3 is characterized in that: described kit also comprises quality controlled serum.
5, kit according to claim 1 and 2 is characterized in that: described crucian carp Vtg polyclonal antibody is a crucian carp Vtg rabbit polyclonal antibody.
6, kit according to claim 1 and 2 is characterized in that: described crucian carp Vtg monoclonal antibody is a crucian carp Vtg mouse resource monoclonal antibody, and it is that sheep anti mouse two is anti-that two of described enzyme labeling resists.
7, a kind of method for preparing the kit of testing environment estrogen effect may further comprise the steps:
1) preparation crucian carp Vtg polyclonal antibody, and bag is sealed with BSA by on ELISA Plate;
2) prepare crucian carp Vtg monoclonal antibody, and mix with equivalent glycerine;
3) preparation resists with two of enzyme labeling;
4) with the crucian carp Vtg polyclonal antibody, the step 2 that obtain in the step 1)) in the crucian carp Vtg monoclonal antibody that obtains, two of the usefulness enzyme labeling that obtains in the step 3) resists, oxygenant and developer are packed jointly, obtain the kit of testing environment estrogen effect.
8, method according to claim 7 is characterized in that: the kit that also original packing of normal concentration crucian carp livetin is advanced described testing environment estrogen effect.
9, according to claim 7 or 8 described methods, it is characterized in that: the kit of also quality controlled serum being packed into described testing environment estrogen effect.
10, method according to claim 7 is characterized in that: described crucian carp Vtg polyclonal antibody is a crucian carp Vtg rabbit polyclonal antibody; Described crucian carp Vtg monoclonal antibody is a crucian carp Vtg mouse resource monoclonal antibody, and two anti-sheep anti mouses two for horseradish peroxidase-labeled of described enzyme labeling are anti-.
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| CN 200310115782 CN1285912C (en) | 2003-12-02 | 2003-12-02 | Kit for detecting environmente female hormone effect and its preparation method |
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|---|---|---|---|
| CN 200310115782 CN1285912C (en) | 2003-12-02 | 2003-12-02 | Kit for detecting environmente female hormone effect and its preparation method |
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| CN1624483A CN1624483A (en) | 2005-06-08 |
| CN1285912C true CN1285912C (en) | 2006-11-22 |
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| CN1904048B (en) * | 2005-07-26 | 2010-11-10 | 复旦大学 | Zebra fish egg yolk protein origin 1 gene regulating and controlling sequence |
| CN100404553C (en) * | 2005-10-11 | 2008-07-23 | 中国水产科学研究院黑龙江水产研究所 | Sturgeon family fish ovovitellin preparation method and uses |
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