CN101738477A - Androgen receptor in-vitro receptor and ligand binding effect-based ELISA quantitative detection method for environmental androgen - Google Patents
Androgen receptor in-vitro receptor and ligand binding effect-based ELISA quantitative detection method for environmental androgen Download PDFInfo
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Abstract
The invention relates to an androgen receptor in-vitro receptor and ligand binding effect-based ELISA quantitative detection method for an environmental androgen, which comprises the following steps: after binding the androgen receptor and the environmental androgen, performing dimerization allosterism on the androgen receptor; binding a bio-labeled DNA probe containing a core sequence of 5'-GGAACAnnnTGTATC-3' of an androgen response element to form a biotin DNA probe-androgen receptor-environmental androgen compound; and adopting a monoclonal antibody of the androgen receptor to absorb and separate the compound; and then performing ELISA detection by a streptavidin-labeled horse radish peroxidase so as to evaluate the effect and content of androgens in a sample. The method has the advantages of simple, convenient and quick operation, high sensitivity, and detection limit of pM level, and can be widely applied to foods, environmental detection, and other fields.
Description
Technical field
The present invention relates to environmental classes androgen detection method, be specifically related to the androgenic ELISA quantitative detecting method of a kind of environmental classes based on androgen receptor extracorporeal receptor-ligand binding effect.
Technical background
In recent years, along with the industrial pollution aggravation, the number of chemical composition of many artificial industrial chemistry materials and agricultural chemicals has the effect of parahormone or antibody internal hormone, may disturb the mankind and wildlife body endocrine normal function, their potential threats comprise a plurality of systems such as the endocrine disturbance that cause the human and animal, it is unusual to grow, inborn defect, fecundity descend, problems such as increase take place tumour, are one of current toxicologic study focuses.
Environmental classes androgen (environmental antiandrogen, EA) the important environment incretion interferent matter of a class that comes to this, but natural androgen in their analogue body, with androgen receptor (androgenreceptor, AR) competitive combination, the interior androgen of prevention body combines with AR's, serve as the AR antagonist, thereby inhibition androgenic activity, performance class androgenic effect, the animal embryo phase exposes and can cause the incomplete or disappearance (particularly epididymis) of reproductive development, external genital organs deformity (hypospadia), cryptorchidism, anogenital waits genotoxicity and deformity apart from shortening.The EA of report relates generally to some non-steroidal drugs such as Flutamide (flutamide at present, FLU), Finasteride (finasteride, FIN), pesticide in the agricultural chemicals, germifuge and herbicide and metabolic product thereof, metabolic product p as pyrethroids (pyrethroids), DDT, p '-DDE, methoxychlor (methoxychlor), sterilization profit (procymidone), vinclozolin (vinclozolin, V) and metabolic product M
1And M
2Deng.
In traditional detection method many with GC and GC-MS method as the standard method that detects EA.But because EA exists with the form of trace potpourri usually, and chemical property is widely different, adopt traditional extracting and enriching, high performance liquid chromatography/mass spectrum/methods such as gas chromatography to isolate single component and measure content, its analysis means complexity, complex operation, waste time and energy, apparatus expensive, need special advanced training, and be difficult to reach the parsing fully of all trace constituents.And exist a large amount of chemicals in the environment, by the end of on November 24th, 2009, at (the Chemical Abstracts Service of CAS, CAS) Deng Ji chemical substance reaches 51 045 390 kinds, in the face of so many chemicals, traditional test method can't satisfy the demands, so must explore high-throughput screening method.U.S. EPA (U.S.EPA) release " environment incretion interferent screening program " in 1998 proposes the layer-stepping screening technique, comprises classification (sorting), priority settings (prioritysetting), first order screening (T
1S) and the second level detect (T
2S) the quadravalence section is comprising T
13 of S stage external and 5 in vivo studies and T
25 living animals in S stage expose experiment.
But so perfect screening program is cost to sacrifice countless animal subject life, and need expends lot of manpower and material resources and time.Therefore, many scholars are devoted to explore quicker, convenient and perfect high throughput settings hormone measurement system, and these trials comprise that acellular (cell-free) and intact cell (wholecell) receptor binding assays, androgen dependent cells proliferation test, androgen rely on genetic transcription activation test, recombinant gene yeast measurement system, GFP-AR immunocytochemistry etc.But, because the limitation of these methods itself, can not accurate quantification etc. as the instability of cellular incubation, cell, make that these method flux are low, the cycle is grown, systematic error is bigger, and the EA in the sample also may be by cell degradation, and accuracy, stability and sensitivity are undesirable.
Existing studies show that, AR is a kind of trans transcript regutation protein of ligand-dependent type, belongs to the nuclear receptor superfamily member.The AR gene generally is made up of 4 domains: low conservative aminoterminal domain (N-Terminusdomain, NTD), high conservative DNA binding structural domain (DNA binding domin, DBD), hinge area (flexible hinge) and high conservative c-terminus ligand binding domains (ligand bindingdomain, LBD), see accompanying drawing 1.Wherein the DBD district can with androgen response element (androgenresponse element, ARE) specific bond on the target gene; The LBD district is in conjunction with hormone, in a single day AR combines with androgen, will activate allosteric and form dimer, combines with the ARE specificity then, stimulates target gene to transcribe, thereby shows various physiological effects.The core sequence of ARE is 5 '-GGAACAnnnTGTATC-3 ' (n is any nucleotide).
Environmental classes androgen EA be exactly with endogenous androgen such as testosterone (Testosterone, T) and protona (Dihydrotestosterone, DHT) similarly form combines with AR, and mediate by AR the same with endogenous androgen produces corresponding pathologic effect.At present based on the screening technique of receptors ligand combination, both can reflect the binding capacity of EA, class androgen sample that again can the concentrated expression EA toxic equivalent (TEQ) that adds up, and be easy to realize high flux is an EA high flux screening and the new direction of monitoring recent years.(GaidoK W such as Gaido, etal.Toxicol Appl Pharmacol, 1997,143 (1): 205-212) yeast cells that adopts recombination yeast measurement system evaluation environmental classes androgen to induce is interior in conjunction with effect, but the method detects in μ M～nM level, and sensitivity and accuracy are still undesirable.
In sum, for numerous in the environment, increase the environmental classes androgen in the compound rapidly, present environmental pollutant analysis Progress in technique also can not satisfy the monitoring requirement far away, and development fast, high flux, cheap triage techniques highly sensitive and good reproducibility be a problem demanding prompt solution.
Summary of the invention
The purpose of this invention is to provide a kind of cheapness, the androgenic ELISA quantitative detecting method of quick, high-throughout environmental classes based on androgen receptor extracorporeal receptor-ligand binding effect.
The present invention utilizes efficient, the specificity of ligand-receptor biology activation effect, and the sensitivity of the enlarge-effect of biotin-avidin and the colour developing of horseradish peroxidase catalytic substrate, set up the add up fast quantification detection technique of toxic equivalent (TEQ) of trace amount environment class androgen (EA), realize the cheapness of trace amount environment class androgen TEQ, fast, high-throughout monitoring.
This purpose of the present invention is to realize by following technical scheme: promptly trace amount environment class androgen (EA) as part with after androgen receptor (AR) combines, acceptor allosteric dimerization, identification and in conjunction with contain androgen response element (ARE) core sequence 5 '-the dna double chain probe of GGAACAnnnTGTATC-3 ' (n is any nucleotide), 5 of this dna probe normal chain ' end biotin (Biotin) mark, form biotin dna probe-AR-EA compound (Biotin-DNA AR-EA probe complex, also be BARC), adopt the special monoclonal antibody absorption of AR and separate BARC, the unconjugated dna probe of flush away, biotin labeled DNA adopts the horseradish peroxidase of streptavidin mark to carry out the ELISA detection among the BARC at combination then, thus the biological effect of EA in the assessment sample.
Therefore, the androgenic ELISA quantitative detecting method of a kind of environmental classes based on androgen receptor extracorporeal receptor-ligand binding effect that the present invention relates to may further comprise the steps:
(1) the synthetic 5 ' end of design is used DNA probe labeled with biotin normal chain, contain in its complete sequence androgen response element core sequence 5 '-GGAACAnnnTGTATC-3 ', wherein n is any nucleotide;
(2) base complementrity of the described probe of synthesis step (1) connects, and equivalent adds (1) described probe, anneals formation 5 ' hold with biotin labeled dna double chain probe in annealing buffer;
(3) androgen receptor is special mouse monoclonal antibody McAb joins in the magnetic bead of sheep anti-mouse igg bag quilt, and washing obtains McAb-magnetic bead solid phase carrier;
(4) will contain the 4 ℃ of joltings in binding buffer liquid of androgenic sample to be checked of environmental classes and androgen receptor hatched 1 hour, hatched 30 minutes for 37 ℃ then, the environmental classes androgen is combined with androgen receptor, acceptor generation allosteric, expose the receptor dna binding structural domain, form androgen receptor-environmental classes androgen compound;
(5) in step (4) compound, add step (2) described 5 ' end is with biotin labeled dna double chain probe, 37 ℃ of reactions 10 minutes, formation biotin dna probe-androgen receptor-environmental classes androgen compound;
(6) the described McAb-magnetic bead of step (3) solid phase carrier will be joined in step (5) gained biotin dna probe-androgen receptor-environmental classes androgen complex solution, form androgen receptor-McAb-magnetic bead IgG compound, with this compound of magnetic force frame absorptive collection, the unconjugated probe of flush away;
(7) horseradish peroxidase of adding streptavidin mark in the described compound of step (6) was hatched 0.5-1 hour for 37 ℃, and the biotin of mark on the dna probe is combined with the streptavidin specificity, and the magnetic force frame is collected magnetic bead compound, the unconjugated enzyme of flush away;
(8) the catalytic substrate tetramethyl benzidine and the hydrogen acceptor hydrogen peroxide of adding horseradish peroxidase in step (7) magnetic bead compound were hatched 10-30 minute for 37 ℃, the catalytic substrate colour developing;
(9) add the stop buffer color development stopping reaction that contains sulfuric acid in the mixed liquor that step (8) has developed the color, magnetic force frame absorption magnetic bead is collected supernatant;
(10) supernatant that step (9) is collected joins the polystyrene ELISA Plate, it also is elisa plate, on the ELISA detector, in the 450nm place, measure each hole absorbance, with be the typical curve comparison that standard items are done with testosterone or protona, determine the accumulative total equivalent of class androgen material in the testing sample.
The advantage of present technique is: (1) is compared with the GC-MS method with traditional GC, and present technique is not identified the difficulty of complex mixture, because the mimic hormone biological process, both can reflect the binding capacity of EA, can reflect the comprehensive androgen sample TEQ activity of EA again; GC and the GC-MS analysis operation is loaded down with trivial details, technical requirement is high, cost is expensive, waste time and energy.(2) compare with genetic transcription activation, cell proliferation experiment, present technique does not have the cell culture period that reached for 2 weeks, and it is extremely short to provide result, test period in 1-2 days.(3) compare in conjunction with detection technique with existing receptors ligand, extracorporeal receptor-the ligand binding effect of present technique by AR and the enlarge-effect by biotin-avidin, it is high to make that sensitivity improves, and meets or exceeds GC and GC-MS analytical chemistry method, detects to reach the pM level.(4) the technology of the present invention is easy and simple to handle, quick, and technology platform is less demanding, and common lab can be carried out, and can be widely used in fields such as food, environment measuring.
Description of drawings
Accompanying drawing 1 is an androgen receptor AR structural representation;
Accompanying drawing 2 has shown that various androgens/class androgen and AR's combines dose-effect curve (binding ability).
Embodiment
The following examples are intended to illustrate implements the embodiment that shows known the best of the present invention, but should not be considered as the present invention is confined to these embodiment.
The synthetic DNA probe labeled with biotin that can combine of embodiment 1 design with the AR specificity
Synthetic 5 ' the end DNA probe labeled with biotin normal chain of design, contain in its complete sequence androgen response element core sequence 5 '-GGAACAnnnTGTATC-3 ', wherein n is any nucleotide, its total order is classified as: 5 '-Biotin-AGACCTACTCTGGAGGAACATATTGTATCGATTGTCCTTGACAGTAAAC GGAACATATTGTATCAAATCTGTTGT-3 ', nucleotide around the core sequence can adjust, and represents with SEQ No1; Synthetic its complementary strand: 5 '-ACAACAGATTTGATACAATATGTTCCGTTTACTGTCAAGGACAATCGATACAATAT GTTCCTCCAGAGTAGGTCT-3 ', represent with SEQ No2; Sequence is by Shanghai bioengineering company limited mark and synthetic.
The synthetic DNA probe labeled with biotin that can combine of embodiment 2 designs with the AR specificity
Synthetic 5 ' end DNA probe labeled with biotin the normal chain of design, contain in its complete sequence androgen response element core sequence 5 '-GGAACAnnnTGTATC-3 ', wherein n is any nucleotide, its total order is classified as: 5 '-Biotin-AGACCTACTCTGGAGGAACATATTGTATCGATTGTCCTTGACAGTAAAC GGAACATATTGTATCAAATCTGTTGT-3 ', nucleotide around the core sequence can adjust, and represents with SEQ No3; Synthetic its complementary strand: 5 '-ACAACAGATTTGATACAATATGTTCCGTTTACTGTCAAGGACAATCGATACAATAT GTTCCTCCAGAGTAGGTCT-3 ', represent with SEQ No4; Sequence is by Shanghai bioengineering company limited mark and synthetic.
(1mM EDTA pH=8.0) dissolves embodiment 1 described SEQ No1 and SEQ No2 for 10mM Tris-HCl, 50mM NaCl, and making its final concentration is 20～100umol/L with annealing buffer.Mole such as two chains is mixed, in 94 ℃ of insulations after 5 minutes, 45 ℃ of insulations 5 minutes, be cooled to room temperature ,-20 ℃ of preservations are standby.
The PBS pre-service that contains 5g/L bovine serum albumin(BSA) and 1mg/ml salmon sperm dna with 300 μ L is coated with the immune magnetic strain of two anti--sheep anti-mouse iggs, in 4 ℃ of 30 rev/mins of joltings 6～12 hours gently on a rotation platform, to reduce non-specific adsorption.Collect magnetic bead with the magnetic force frame, with containing the cold PBS washing magnetic bead of 5g/L BSA 3 times, standby again.Mouse source property AR monoclonal antibody McAb and pretreated immunomagnetic beads with dilution in 1: 300 slightly shook under room temperature 1～3 hour then, formed magnetic bead-two and resisted-the McAb solid phase carrier.The magnetic force frame is collected this solid phase carrier, washs 3 times, and is standby.
The external specific bond system of embodiment 5 trace EA and AR
In the sample EA and AR be combined in following binding buffer liquid system and condition is carried out: 50mMTris-HCl, 1mM EDTA, 500mM KCl, 2mM dithioerythritol (DTT), 2mg/ml bovine serum albumin(BSA) (BSA), 5% glycerine, binding buffer liquid pH 7.6.Adopt 100 μ L systems, hatch a few hours for 4 ℃, move to 37 ℃ and hatched 30 minutes.
The experiment process that combines of embodiment 6 EA and AR
Final concentration is the AR 50 μ L of 10nM and the androgen/class androgen of 50 μ L series concentration, and (final concentration is from 1.0 * 10 as testosterone, protona, Flutamide, vinclozolin etc.
-14～1.0 * 10
-6M), in embodiment 5 described binding buffer liquid, hatched 1 hour for 4 ℃, moving to 37 ℃ hatched 30 minutes, promote the Dimerized allosteric of AR, expose the receptor dna binding structural domain, adding 5ng embodiment 3 described dna double chain probes and hatched 10 minutes, form biotin dna probe-AR-EA compound, also is BARC.Do not add androgen as blank, all are tried sample standard deviation and are done multiple hole.With this BARC add embodiment 4 described magnetic beads-two anti--the McAb solid phase carrier, mixing gently, room temperature, 30 rev/mins of jogs are 1 hour on a rotation platform, promptly get BARC-McAb-magnetic bead IgG compound, with this compound of magnetic force frame adsorptive separation, remove supernatant, the probe that flush away is free.
The Avidin of embodiment 7 bonding probes amplifies ELISA and detects
In the magnetic bead compound that embodiment 6 collects, the horseradish peroxidase that adds the streptavidin mark, hatched 0.5～1 hour for 37 ℃, the biotin of mark on the dna probe is combined with the streptavidin specificity, form compound, in compound, add the catalytic substrate tetramethyl benzidine TMB and the hydrogen acceptor H of horseradish peroxidase
2O
2, to hatch 10～30 minutes for 37 ℃, catalysis TMB colour developing adds the stop buffer color development stopping reaction that contains sulfuric acid then, and the magnetic force frame is collected the supernatant that developed the color.This supernatant is joined the polystyrene ELISA Plate, it also is elisa plate, with this elisa plate on the ELISA detector, in the 450nm place, measure each hole absorbance, also being the OD value, and is the typical curve comparison that standard items are done with series concentration protona (DHT) or testosterone (T), determines the relative content and the accumulative total equivalent of class androgen material in the testing sample.The concentration of series standard product can be divided into: 1 * 10
-12, 1 * 10
-11, 1 * 10
-10, 1 * 10
-9, 1 * 10
-8, 1 * 10
-7, 1 * 10
-6, 1 * 10
-5M.
The binding ability of embodiment 8 observation different androgens/class androgens and AR and detection architecture are to the detectability of trace EA
The androgen and the class androgen standard items of preparation variable concentrations, comprise testosterone (T), protona (DHT), alpha..alpha..alpha.-Trifluoro-2-methyl-4'-nitro-m-lactotoluidide (Hydroxyflutamide, OH-FLU), progestational hormone (Progesterone, Pro), p, p '-DDE etc., in the articulated system of embodiment 6～7, observe the binding ability of different androgens/class androgen and AR, and measure the sensitivity (detection lower limit) and the accuracy (recovery) of detection architecture.The result shows that binding ability is DHT ≈ T＞Pro＞OH-FLU＞p in proper order, and p '-DDE sees accompanying drawing 2.With conventional cell proliferation experiment and GC/MS analysis result is contrast method, compares.Present technique can be provided quantitative result in 1-2 days, sensitivity can reach the pM level, compared with classic method, and this method has fast, efficient, high flux and accuracy, highly sensitive advantage.
The nucleotide sequence relevant with the present invention:
＜110〉The First Affiliated Hospital of Third Military Medical University of PLA
＜120〉the androgenic ELISA quantitative detecting method of a kind of environmental classes based on androgen receptor extracorporeal receptor-ligand binding effect
<160>4
<170>
<210>1
<211>75
<212>DNA
＜213〉artificial sequence
<220>
＜221〉bonding probes normal chain
＜223〉design according to the androgen response element, contain two androgen response elements, its 5 ' end is used biotin labeling, as the distinguished sequence in conjunction with androgen receptor.
<400>1
5′-Biotin-AGACCTACTCTGGAGGAACATATTGTATCGATTGTCCTTGACAGTAAACGGAACATATTGTATCAAATCTGTTGT-3′
<210>2
<211>75
<212>DNA
＜213〉artificial sequence
<220>
＜221〉bonding probes complementary strand
＜223〉be the complementary strand of SEQ No1
<400>2
5′-ACAACAGATTTGATACAATATGTTCCGTTTACTGTCAAGGACAATCGATACAATATGTTCCTCCAGAGTAGGTCT-3′
<210>3
<211>75
<212>DNA
＜213〉artificial sequence
<220>
＜221〉bonding probes normal chain
＜223〉design according to the androgen response element, contain two androgen response elements, its 5 ' end is used biotin labeling, as the distinguished sequence in conjunction with androgen receptor.
<400>3
5′-Biotin-GGCACTACTCTGGAGGAACACGGTGTATCGATTGTCCTTGACAGTAAACGGAACACGGTGTATCACATCTGCCTA-3′
<210>4
<211>75
<212>DNA
＜213〉artificial sequence
<220>
＜221〉bonding probes complementary strand
＜223〉be the complementary strand of SEQ No3
<400>4
5′-TAGGCAGATGTGATACACCGTGTTCCGTTTACTGTCAAGGACAATCGATACAACCGGTTCCTCCAGAGTAGTGCC-3′
Claims (3)
1. androgenic ELISA quantitative detecting method of the environmental classes based on androgen receptor extracorporeal receptor-ligand binding effect is characterized in that: may further comprise the steps:
(1) the synthetic 5 ' end of design is used DNA probe labeled with biotin normal chain, contain in its complete sequence androgen response element core sequence 5 '-GGAACAnnnTGTATC-3 ', wherein n is any nucleotide;
(2) base complementrity of the described probe of synthesis step (1) connects, and equivalent adds (1) described probe, anneals in annealing buffer, forms 5 ' and holds with biotin labeled dna double chain probe;
(3) androgen receptor is special mouse monoclonal antibody McAb joins in the magnetic bead of sheep anti-mouse igg bag quilt, and washing obtains McAb-magnetic bead solid phase carrier;
(4) will contain the 4 ℃ of joltings in binding buffer liquid of androgenic sample to be checked of environmental classes and androgen receptor hatched 1 hour, hatched 30 minutes for 37 ℃ then, the environmental classes androgen is combined with androgen receptor, acceptor generation allosteric, expose the receptor dna binding structural domain, form androgen receptor-environmental classes androgen compound;
(5) in step (4) compound, add step (2) described 5 ' end is with biotin labeled dna double chain probe, 37 ℃ of reactions 10 minutes, formation biotin dna probe-androgen receptor-environmental classes androgen compound;
(6) the described McAb-magnetic bead of step (3) solid phase carrier will be joined in step (5) gained biotin dna probe-androgen receptor-environmental classes androgen complex solution, form androgen receptor-McAb-magnetic bead IgG compound, with this compound of magnetic force frame absorptive collection, the unconjugated probe of flush away;
(7) horseradish peroxidase of adding streptavidin mark in the described compound of step (6) was hatched 0.5-1 hour for 37 ℃, and the biotin of mark on the dna probe is combined with the streptavidin specificity, and the magnetic force frame is collected magnetic bead compound, the unconjugated enzyme of flush away;
(8) the catalytic substrate tetramethyl benzidine and the hydrogen acceptor hydrogen peroxide of adding horseradish peroxidase in step (7) magnetic bead compound were hatched 10-30 minute for 37 ℃, the catalytic substrate colour developing;
(9) add the stop buffer color development stopping reaction that contains sulfuric acid in the mixed liquor that step (8) has developed the color, magnetic force frame absorption magnetic bead is collected supernatant;
(10) supernatant that step (9) is collected joins the polystyrene ELISA Plate, it also is elisa plate, on the ELISA detector, in the 450nm place, measure each hole absorbance, with be the typical curve comparison that standard items are done with testosterone or protona, determine the accumulative total equivalent of class androgen material in the testing sample.
2. the androgenic ELISA quantitative detecting method of a kind of environmental classes according to claim 1 based on androgen receptor extracorporeal receptor-ligand binding effect, it is characterized in that: every liter of the binding buffer liquid in the step (4) is by 1.2～12.5g Tris-HCl, 0.1～1.5g EDTA, 7.5～35g KCl, 0.1～2g dithioerythritol, 1～5g bovine serum albumin(BSA), 50～100ml glycerine and surplus are that deionized water is formed.
3. the androgenic ELISA quantitative detecting method of a kind of environmental classes based on androgen receptor extracorporeal receptor-ligand binding effect according to claim 1 is characterized in that: 5 ' end is classified SEQ No1 or SEQ No3 as with the nucleotides sequence of DNA probe labeled with biotin normal chain.
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| CN111175489A (en) * | 2020-01-03 | 2020-05-19 | 山西大学 | Method for evaluating blocking activity of molecular blocking antibody of immune checkpoint by magnetic beads |
| WO2023087942A1 (en) * | 2021-11-18 | 2023-05-25 | 上海北昂医药科技股份有限公司 | Delisa sample capable of increasing proportion of effective microbeads, and preparation and detection method therefor |
| CN114437202A (en) * | 2022-01-18 | 2022-05-06 | 四川大学华西医院 | Androgen response element and application thereof in detecting environmental androgen |
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