WO2002025278A2 - Verfahren zur bestimmung der peptidhormon-aktivitäten oder der steroidhormon-aktivitäten eines materials oder stoffgemisches - Google Patents
Verfahren zur bestimmung der peptidhormon-aktivitäten oder der steroidhormon-aktivitäten eines materials oder stoffgemisches Download PDFInfo
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- WO2002025278A2 WO2002025278A2 PCT/DE2001/003628 DE0103628W WO0225278A2 WO 2002025278 A2 WO2002025278 A2 WO 2002025278A2 DE 0103628 W DE0103628 W DE 0103628W WO 0225278 A2 WO0225278 A2 WO 0225278A2
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- 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
- G01N33/743—Steroid hormones
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- 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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
Definitions
- the invention relates to a method for the qualification and quantification of hormonal and antagonistic hormonal activities of a material, a bioassay, and the use thereof.
- Hormones are bioactive signaling molecules that are synthesized in specialized, so-called endocrine cells of every organism, usually. are released into the blood and trigger certain biological effects by reversibly binding to receptors of target cells in a highly affine and highly specific manner; this sends the hormone signal into the cell.
- the peptide hormones e.g. insulin, thyroid hormones, adrenaline
- the steroid hormones which mainly include the glucocorticoids, mineralocorticoids, androgens (incl. Androgenic anabolics, "doping agents"), estrogens and gestagens, are the most important hormones for almost all living beings. They regulate growth, physical education and are indispensable for the process and maintenance of vital physiological and biological functions, e.g. of reproduction.
- steroids are used in a not inconsiderable amount in animal fattening - it is therefore also important to be able to quickly and easily determine whether these mostly illicit means have been used and then end up in human consumption.
- the decisive factor in determining a so-called hormone status and, if necessary, its dynamics and the resulting diagnostic and therapeutic consequences is not the amount or concentration of the respective peptide or steroid hormones, but rather their activity, i.e. the type and extent of the biological effect, which, for example is influenced by the dissociation constants of the ligand-receptor complex, but also by many other factors, such as the presence of transport proteins, etc.
- the principle of action of the steroid hormones will first be explained. These mediate their effect significantly via receptors specific for the respective steroid hormone classes, which according to the prevailing doctrine are located within the target cells.
- the receptors for peptide hormones are located on or in the cell membrane, i.e. the cell outer shell). They form a very binding-resistant receptor-steroid complex. This complex formation is an essential link in the hormone-dependent signal chain, which leads to the hormone-specific effect (see below).
- steroid hormone receptors examples include the glucocorticoid receptor (GR), which binds the glucocorticoid cortisol; the androgen receptor (AR), which binds the male sex hormone testosterone and various synthetic steroid hormone analogues, such as anabolic steroids; the estrogen receptors - ER ⁇ and ERß - which bind the female sex hormone estradiol, but also synthetic estrogens (e.g.
- HRE hormone responsive elements
- the steroid hormone cortisol in the liver cell the formation of the essential enzyme tyrosine aminotransferase; estradiol that of PR or that of transcortin (indispensable transport protein for glucocorticoids in the blood).
- the object is achieved by a method with the features of claim 1.
- Advantageous further developments result from the dependent claims.
- the invention also relates to a determination assay for the hormone activities of materials according to claim 9.
- a suitable cell either a) with endogenous hormone receptor that is transient or stable with a recombinant hormone-sensitive reporter gene construct (reporter expression plasmid), or additionally with a recombinant specific hormone receptor expression plasmid mono- or is co-transfected, or b) without endogenous hormone receptor, which is transiently or stably co-transfected with a recombinant hormone-sensitive reporter gene construct and a recombinant specific hormone receptor expression plasmid, which in case a) or b ) - as a transfected cell can express a signaling, easily measurable product.
- reporter expression plasmid reporter expression plasmid
- transient transfection means the introduction of foreign DNA into the cell, either into its cytoplasm only for the duration of a cell cycle
- stable transfection means the permanent introduction of foreign DNA into its genome.
- Genetically modified (recombinant) plasmids [AA1] ("cloning vectors"), which contain the genetic building instructions for the desired reporter protein or the specific hormone receptor, generally serve as vehicles. These genes are preceded by a specific promoter.
- This can either be a constitutive promoter (eg with SV40 or Tk (thymidine kinase) in the receptor expression plasmid), the permanent expression, or an inducible promoter (eg with HRE in the reporter expression plasmid), the stimulable (eg by a hormone ) Expression of the downstream (foreign) gene causes.
- a constitutive promoter eg with SV40 or Tk (thymidine kinase) in the receptor expression plasmid
- the permanent expression eg with HRE in the reporter expression plasmid
- an inducible promoter eg with HRE in the reporter expression plasmid
- the stimulable eg by a hormone
- the most effective transfection method is the so-called “non-recombinant adenovirus polylysine" technique.
- Sommer B. et al . “Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complexes", Calcif. Tissue Int., 64 (1999), 45-49. This method is particularly indicated for the transfection of differentiated eukaryotic cells with endogenous hormone receptor according to point 1.
- the hormone-active component (s) of the material forms after introduction into the transfected cell - (for example by adding the material to the cell culture medium) - with the endogenous and / or recombinant steroid receptor (for example glucocorticoid receptor, estrogen receptor, androgen receptor) , Mineralocorticoid receptor, progesterone receptor, etc.) a binding-resistant ligand-receptor complex.
- ligand forms after introduction into the transfected cell - (for example by adding the material to the cell culture medium) - with the endogenous and / or recombinant steroid receptor (for example glucocorticoid receptor, estrogen receptor, androgen receptor) , Mineralocorticoid receptor, progesterone receptor, etc.) a binding-resistant ligand-receptor complex.
- reporter proteins are the enzymes luciferase and ⁇ -galactosidase or green, red, yellow fluorescent proteins (GFP, RFP, YFP), which can easily be prepared by existing devices, using established protocols and with commercially available kits, luminescence reaction in the luminometer or fluorescence reaction in FACS (Fluorescent Activated Cell Sorting) can be quantified.
- the "Dual-Luciferase TM Reporter Assay System” available from Promega, Madison, Wl, USA; cat. No. E1916
- luciferase measurement of the successfully transfected cells by measuring the constitutively expressed Renilla luciferase.
- FACS Fluorescent Activated Cell Sorting
- Some cells with endogenous hormone receptor according to 1. contain different endogenous hormone receptors: HepG2 and CC-1, HTC and other "hepatoma tissue cells" (non-hepatoma or hepatoma lines of human or rat liver), for example, contain glucocorticoid receptor and estrogen receptor; MCF-7 (human breast cancer cell line) even estrogen receptor, glucocorticoid receptor and androgen receptor.
- the material to be examined should be discriminated between different qualities of steroid hormone activities, for example glucocorticoid, estrogen, androgen, mineralocorticoid or Gestagen activity, etc., therefore cells without endogenous hormone receptor according to 1.b) are used; these are expressed with the respective hormone receptor expression plasmid, i.e.
- the appropriate hormone-sensitive Reporter gene plasmid for all listed activity qualities, with the exception of the estrogen-active materials, is, for example, GRE ("glucocorticoid responsible elements") - Luc with a GRE2-TATA promoter which codes for luciferase; this plasmid (pLCO546A) - also generally referred to as a vector - reacts significantly more sensitively and therefore more strongly to inducing substances with hormone activity as the commonly used plasmid with an MMTV-GRE promoter.
- GRE glucocorticoid responsible elements
- pLCO546A this plasmid (pLCO546A) - also generally referred to as a vector - reacts significantly more sensitively and therefore more strongly to inducing substances with hormone activity as the commonly used plasmid with an MMTV-GRE promoter.
- Suitable for estrogen-active materials is, for example, ERE-Luc with an ERE-TATA promoter which also codes for luciferase.
- Suitable cells / cell lines as such are, for example, CV-1 and COS-1, COS-7 (monkey kidney cells), and above all special yeast cell lines which have been shown to express various gene products, including various steroid like receptors ", have proven very successful (McEwan IJ:" Investigation of steroid receptor function in the budding yeast Saccharomyces cerevisiae ", FEMS Microbiol. Lett. 179 (1999), 183-4). All listed and other suitable cell lines, as under point 1. a) and 1. b) are commercially available from various cell banks and with a precise definition; some important ones are listed as examples:
- DSMZ Human and Animal Cell Cultures
- German Collection of Microorganisms & Cell Cultures Mascheroder Weg 1b, Braunschweig, D-38124, Germany
- yeast cells unlike eukaryotic cells, have a cell wall, the above-mentioned transfection technique is not suitable.
- the person skilled in the art is also familiar with processes, for example the lithium acetate method. (Gietz RD et "Studies on the transformation of intact yeast cells by the LiAc / SS-DNA / PEG procedure", Yeast, 11 (1995), 355-60.
- Co-transfection of cells (see point 1. a) with a hormone receptor expression plasmid considerably increases the measuring sensitivity of the transfected cell: namely, in addition to the naturally existing ("endogenous") steroid receptors, other, “recombinant” receptors ", that is Nature-identical, made. As a result, the cells respond to even lower concentrations of hormone-active substances and thus represent a particularly sensitive measuring arrangement.
- Co-transfection i.d.S. is therefore an essential element of the invention.
- bioassay according to the invention for the qualification and quantification of substances / molecules and other materials with respect to, for example, suspected steroid hormone ac- Activity, steroid hormone agonism and possibly antagonism is also advantageous for another reason and is superior to the usual one for determining the amount of substance: as already mentioned above, there are numerous naturally occurring substances and those from the technical environment, or chemical compounds and their often unknown Metabolites with steroid hormone activity (e.g. phyto-estrogens, pesticides and DDT and its still ubiquitous chemical derivatives that act like estrogens). Possibly common and important uses of the invention include that of determining:
- Estrogen-active individual materials and / or mixtures of substances of physiological origin e.g. in the serum of humans and cattle
- foods, cosmetics and other pharmaceutical products as well as in some technical-synthetic "Permanent Organic Pollutants” (POP).
- POP Permanent Organic Pollutants
- COS-1, COS-7, HeLa undifferentiated, aneuploid cell line from a rapidly growing human cervical carcinoma
- MCF-7 CHO ("Chinese Chineseamster ovary cells") cells, in particular Yeast cell lines (see above) which are co-transfected with an androgen receptor or estrogen receptor-coding expression plasmid and a luciferase reporter gene construct which contains a GRE2-TATA or ERE-TATA promoter, the principle of application and implementation of the assay are as described above.
- Fig. 1 Androgen-induced luciferase activity in human genital skin fibroblasts (GHF) in culture, which with three different luciferase gene constructs of different Different androgen sensitivity (“Luc”) were transfected: PRE2-Tk-Luc, MMTV-Luc, GRE2-TATA-Luc
- Fig. 2 Dose-effect relationship of a steroid hormone with known glucocorticoid (GC) activity: corticosterone-induced luciferase activity in CC1 cells without (o) and with ( ⁇ ) co-expression of the glucocorticoid receptor
- Fig. 4 Induction-time-dependent dose-effect relationship of a supposedly inactive steroid hormone: 21-hemisuccinate corticosterone: BSA-induced luciferase activity in CC1 cells
- Fig. 7 Dose-effect relationship of various steroid hormones with different levels of GC activity: induction of luciferase activity by corticosterone (B), medroxyprogesterone (MP), estrone (E1), progesterone (P) and 17 ⁇ -21-dihydroxy-progesterone (Cortex) in CC1 cells
- Fig. 8 Dose-effect relationship of various steroid hormones with different GC activity: induction of luciferase activity by corticosterone (B), medroxyprogesterone (MP), estrone (E1), progesterone (P) and 17 ⁇ -21-dihydroxy progesterone (Cortex) in MH 3924 cells
- FIG. 9 Antagonistic effect of a synthetic steroid hormone on the GC activity: inhibition of the luciferase activity induced by corticosterone (B) and 21-hemisuccinate corticosterone: BSA (B: BSA) in CC1 cells by RU 486 (mifepristone TM)
- Fig. 10 Dose-effect relationship of GC-active steroid hormones: corticosterone (B) and 21-hemisuccinate-corticosterone: BSA (B: BSA) -induced green fluorescent protein (GFP) activity in MH 3924 cells, stable with an MMTV-GFP reporter gene construct and transiently transfected with a glucocorticoid receptor expression plasmid
- non-recombinant adenovirus polylysine technique was always used as the transient transfection method of the experiments shown in FIGS. 1-10.
- Fig. 11 Dose-effect relationship of a synthetic androgen with anabolic activity: induction of luciferase activity by R1881 (methyltrienolone) in HeLa cells, co-transfected with an androgen receptor expression plasmid.
- the DOTAP method was used as the transfection method (Boehringer, Mannheim, Germany).
- Fig. 12 Estrogen activity determination of E2 and E1 by co-transfected COS-1 cells (Ex. 13). The superfect method from Qiagen, USA was used as the transient transfection method for the COS-1 cells.
- Fig. 13 Androgen activity determination of testosterone with co-transfected COS-1 cells (Ex. 14)
- GHF human genital skin fibroblasts
- Luc luciferase gene constructs
- GRE promoters glucocorticoid-responsive element promoters
- ARE glucocorticoid-responsive elements
- GHF contain only androgen receptors (AR) as steroid hormone receptors.
- the miboleron used in this experiment is a synthetic anabolic androgen.
- CC-1 transiently transfected by means of GRE2-TATA-Luc (these contain nendogenously in addition to ER also FR) or MH 3924 cells in culture are the determination of the glucocorticoid activity of corticosterone and of bovine serum albumin (BSA : "bovine serum albumin") coupled corticosterone derivative (see FIG. 4); these cell lines contain endogenous glucocorticoid receptors.
- BSA bovine serum albumin
- the steroid concentration - here corticosterone concentration - determines the extent of the glucocorticoid activity.
- nM 10 "9 ) means nanoMol / liter. Induction duration: 24 h (see also example 12).
- Example 3 The following examples therefore always relate to transiently co-transfected CC1 or MH 3924 cells.
- Example 3
- the measuring system "co-transfected cell” is so sensitive that materials - here corticosterone - the hormone activity inherent in them either with a long induction time (eg 24 h) even at very low concentrations, or in higher concentrations reveal a very short induction time (eg 10 min).
- FIG. 4a as in FIG. 2a, it is shown how co-transfected CC1 cells are produced by a corticosterone derivative coupled to bovine serum albumin (BSA: "bovine serum albumin”), which is generally considered to be glucocorticoid-inactive, luciferase induce.
- BSA bovine serum albumin
- glucocorticoid activity detected here is not due to the fact that free corticosterone molecules within this formulation are responsible. Thus, even a low glucocorticoid-like activity can be detected by the assay according to the invention. This also results from FIG.
- transfected MH 3924 cells can be used to measure an easily measurable luciferase induction even after a short-term induction of 10 to 30 min with a very low concentration of corticosterone: BSA ( ⁇ 50 nM).
- BSA derivatives classified as inactive 21: BSA, 3: BSA; 200 nM each
- CC1 cells according to Example 4 The difference becomes particularly clear with a long induction time. It can be seen that obviously slight differences in the activity of the two steroid derivatives by means of the assay according to the invention are evident within a relatively short period of time. Mood durations can be determined, as is desired, for example, for "doping" tests or for rapid tests.
- FIG. 7 shows a comparison of the glucocorticoid activity of various steroid hormones at different concentrations (induction time: 24 h).
- Example 6 - are highly active while the natural estrogen estrone (E1) is inactive.
- the natural progestogens (P) and especially cortexolone (17 ⁇ -, 21-dihydroxy progesterone) classified as glucocorticoid antagonistic show significant glucocorticoid activity - see. a. 7a “higher sensitivity of the MH 3924 cells”.
- Example 8 Assay to determine the glucocorticoid activity of various steroid hormones with MH
- Glucocorticoid active hormones This becomes particularly clear for progesterone, but also for corticosterone - s. Fig. 8 and also Fig. 7a.
- RU 38486 known as Mifepristone, the essential component of the so-called “abortion pill”.
- the luciferase-inducing effect (glucocorticoid agonism) of corticosterone and 21-hemisuccinate corticosterone: BSA is largely blocked by RU 38486 - i.e. Luciferase induction is inhibited.
- MH 3924 cells were transiently co-transfected with a glucocorticoid receptor expression plasmid and stably with a GFP reporter gene construct which contains the MMTV-GRE promoter. (Induction time: 24 h). The result is shown in FIG. 10. The considerably lower sensitivity of this reporter gene to GRE2-TATA-Luc is clear from the comparison with FIGS. 3 and 4 (see also FIG.
- FIG. 11 shows the determination of the androgen activity of methyltrienolone (R1881), a synthetic anabolic androgen, by means of luciferase induction in cultured HeLa cells.
- R1881 methyltrienolone
- This androgen receptor-free cell line was transiently co-transfected with GRE2-TATA-Luc and a recombinant androgen receptor expression.
- Induction duration: 24 h .. pM (10 " 12 ) means picoMol / liter.
- GRE2-TATA-Luc pLCO546A
- Luciferase (Luc) gene with a minimal glucocorticoid-inducible GRE promoter GRE-TATA
- GRE-TATA minimal glucocorticoid-inducible GRE promoter
- pRL-TK Vector Renilla luciferase
- HSV-TK herpes simplex virus thymidine kinase
- GR expression plasmid pSTC-rGR: for the GR of the rat with a constitutive cytomegalus virus (CMV) promoter
- the replication-defective adenovirus type 5 (Adv5, strain DL-312) lacks the "early region” genes E1a and E1b.
- the multiplication of the viruses took place in stably transfected human 293 embryonic kidney cells (293 cells) complementing the missing genes.
- poly-L-lysine (pLys; Sigma, St. Louis, MO, USA) was applied to the using the 1-ethyl-3- (dimethyl-aminopropyl) carbodiimide (EDC) technique
- pLys poly-L-lysine
- EDC 1-ethyl-3- (dimethyl-aminopropyl) carbodiimide
- DMEM Dulbecco's Modification of Eagle's Medium; available from Mediatech Inc. under the brand cellgro® Park Center, Herndon, Va, USA
- 10% fetal calf serum is used as the nutrient medium for the cultivation of the cells.
- the cells from confluently overgrown culture dishes are converted 24 hours before the transfection in 12-well culture plates to 2.5 * 10 5 cells per cavity. During this 24 hour period, the cells grow in the medium with steroid hormone-free fetal calf serum. It is transfected when the cells have a cell density of about 60 to 90% confluence.
- the medium is changed after washing with phosphate buffer to 0.5 ml medium without fetal calf serum.
- the transfection solution per batch for one cavity is prepared in polystyrene tubes as follows:
- adenovirus pLys solution with 5.7 * 10 10 particles / ml are mixed and incubated for 30 min at room temperature in the dark. After adding 7.55 ⁇ l of pLys-HBS solution in a weight ratio of DNA to pLys of 1: 1, 3, the mixture is incubated again in the dark at room temperature for 30 min.
- the cells are incubated with 25 ⁇ l of this transfection solution for two hours.
- the transfection medium is then replaced by 1.5 ml of fresh medium.
- 12.5 ⁇ l steroid hormone solution is added to induce luciferase production.
- Blank value approaches receive hormone-free phosphate buffer with 0.1% ethanol.
- the ethanolic stock solutions are evaporated in glass vessels at 50 ° C and 1 atm in a Speed-Vac, resolubilized in 3 ⁇ l ethanol and diluted with phosphate buffer.
- the specified steroid concentrations from 20 to 1000 nM (double values are the final concentrations in the medium.
- the ethanol fraction is not more than 0.1%.
- the control batch (double value) contains no hormone.
- the cells are induction for a maximum of 24 hours exposed to corticosterone. In the case of short-term induction of 10, 30 or 60 min, the steroid-containing medium is replaced by steroid-free medium.
- the cells are 48 hours with a medium- Change and after 24 hours with a second hormone dose for 10, 30 or 60 minutes.
- the cells are lysed with 200 ⁇ l lysis buffer (see following section) per cavity with occasional swirling for 20 min at room temperature.
- the lysed cells are frozen in the culture plates at -80 ° C. until the luciferase activity is measured.
- Luc activity can be measured in any luminometer, e.g. BIOLUMAT LB 95000 T (from Berthold, Wildbad, Black Forest), and is carried out according to the protocol for the "Dual-Luciferase Reporter Assay System” (available from Promega, Madison, Wl, USA).
- the Firefly activity is measured first, and then that of the Renilla luciferase.
- 10 ⁇ l lysate is mixed with 50 ⁇ l LAR buffer, and the light signals are measured after 10fsec in the luminometer.
- the measuring mode is set to 10 sec, 25 ° C, integer.
- 50 ⁇ l of “Stop & Glow” buffer are added, the mixture is vortexed vigorously for 5 seconds and measured again in the luminometer 10 seconds after the addition of the buffer.
- the measurement of the two luciferases is carried out with lysates from cells which have been transfected either only with Firefly or only with Renilla luciferase reporter gene.
- the induction of luciferase synthesis per culture batch is calculated as a multiple of the Firefly pro Renilla luciferase activity of the test batch, based on the corresponding quotient of the control batch without hormones.
- the x-fold induction of each approach is based on that of the approach with 100 nM corticosterone.
- the mean values of Luc (Std) are used for the evaluation and the representations. The final values are the result of the following calculation: 18
- Luc (100) induction of Luc by 100 nM corticosterone in a single experiment, standardized Firefly-Luc / Renilla-Luc
- Luc (N) induction of Luc by unequal 100 nM corticosterone in a single experiment, standardized Firefly-Luc / Renilla-Luc
- Luc ( p. ) Luc values of Firefly-Luc without steroid
- Luc (R +) Luc values of the Renilla-Luc with variable. Steroid (concentration)
- Luc (R-) Luc values of the Renilla-Luc without steroid
- Reporter gene construct ERE-E1A-Luc; Luciferase (Luc) gene with a minimal estrogen-inducible ERE promoter (ERE-TATA)
- Constitutive reporter gene Renilla luciferase (pRL-TK Vector) with the herpes simplex virus thymidine kinase (HSV-TK) promoter (available from Promega, Madison, Wl, USA.)
- ER expression plasmid pSTC-TK-hER: for the estrogen receptor of humans with a constitutive thymidine kinase (TK) promoter
- Estrogen antagonist Tamoxifen as a competitive ER antagonist
- COS and CV cells have no endogenous hormone receptors and therefore, as a prerequisite for performing the boi assay, they must be transient or stable with a recombinant specific hormone receptor expression plasmid. be co-transfected
- the COS-1 cells are cultivated analogously to Example 12
- GRE2-TATA-Luc pLCO546A
- Luciferase (Luc) gene with a minimal glucocorticoid-inducible GRE promoter GRE-TATA
- Renilla luciferase Renilla luciferase (pRL-TK Vector) with the herpes simplex virus thymidine kinase (HSV-TK) promoter (available from Promega, Madison, Wl, USA)
- AR expression plasmid pSTC-hAR: for the AR of humans with a constitutive cytomegalus virus (CMV) promoter
- the entire bioassay is carried out as in Example 13.
- the amounts of DNA used per cavity for the reporter gene construct, the AR expression plasmid and for the constitutive reporter gene pRL-TK are identical to those mentioned there.
- the testosterone concentrations from 100 pM to 10 nM (double values) are the final concentrations in the medium.
- the MH 3924 cell line (source: PD Dr. Doris Mayer, AG Hormone Effect and Signal Transduction, FS Tumor Cell Regulation, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg) is representative of glucocorticoid agonists because of its high sensitivity numerous other hepatoma cell lines of rats and humans (sources of supply e.g. ATCC, DSMZ, ECACC - see above).
- the parameters listed by way of example therefore represent optimization elements for a kit for the use of the bioassay according to the invention for the qualification and quantification of the steroid hormone agonism and possible antagonism of substances / molecules or material mixtures, the skilled person selecting the suitable cell lines and the reporter gene constructs in accordance with the posed Requirements (durability, sensitivity, specificity, etc.) is possible due to his specialist knowledge and in accordance with the teaching of the invention.
- the invention is therefore in no way limited to the examples explained here.
- the above invention is not limited to the exact construction and the exemplary embodiments given, but a wide variety of modifications are possible without deviations from the concept and scope of protection.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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AU2002213808A AU2002213808A1 (en) | 2000-09-20 | 2001-09-20 | Method for determining the peptide hormone activities or the steroid hormone activities of a material or substance mixture |
US10/380,963 US20040023264A1 (en) | 2000-09-20 | 2001-09-20 | Method for determining the peptide hormone activities or the steroid hormone activities of a material or substance mixture |
EP01982131A EP1325327A2 (de) | 2000-09-20 | 2001-09-20 | Verfahren zur bestimmung der peptidhormon-aktivitäten oder der steroidhormon-aktivitäten eines materials oder stoffgemisches |
DE10194024T DE10194024D2 (de) | 2000-09-20 | 2001-09-20 | Verfahren zur Bestimmung der Peptidhormon-Aktivitäten oder der Steroidhormon-Aktivitäten eines Materials oder Stoffgemisches |
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DE10046647.8 | 2000-09-20 | ||
DE10046647A DE10046647A1 (de) | 2000-09-20 | 2000-09-20 | Verfahren zur Bestimmung der Peptidhormon-Aktivitäten oder der Steroidhormon-Aktivitäten eines Materials oder Stoffgemisches |
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CN113801889A (zh) * | 2021-09-18 | 2021-12-17 | 中国农业科学院农业质量标准与检测技术研究所 | 细胞筛选模型及其构建方法和应用、酵母菌及其制备方法和应用 |
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US20070004045A1 (en) * | 2005-06-07 | 2007-01-04 | Xia Xu | Analysis of large numbers of estrogens and other steroids and applications thereof |
US20090215111A1 (en) * | 2005-06-07 | 2009-08-27 | Veenstra Timothy D | Analysis of steroid hormones in thin tissue sections |
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2000
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2001
- 2001-09-20 EP EP01982131A patent/EP1325327A2/de not_active Withdrawn
- 2001-09-20 AU AU2002213808A patent/AU2002213808A1/en not_active Abandoned
- 2001-09-20 US US10/380,963 patent/US20040023264A1/en not_active Abandoned
- 2001-09-20 WO PCT/DE2001/003628 patent/WO2002025278A2/de active Application Filing
- 2001-09-20 DE DE10194024T patent/DE10194024D2/de not_active Expired - Lifetime
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113801889A (zh) * | 2021-09-18 | 2021-12-17 | 中国农业科学院农业质量标准与检测技术研究所 | 细胞筛选模型及其构建方法和应用、酵母菌及其制备方法和应用 |
CN113801889B (zh) * | 2021-09-18 | 2023-04-07 | 中国农业科学院农业质量标准与检测技术研究所 | 细胞筛选模型及其构建方法和应用、酵母菌及其制备方法和应用 |
Also Published As
Publication number | Publication date |
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EP1325327A2 (de) | 2003-07-09 |
DE10194024D2 (de) | 2003-10-02 |
US20040023264A1 (en) | 2004-02-05 |
DE10046647A1 (de) | 2002-03-28 |
AU2002213808A1 (en) | 2002-04-02 |
WO2002025278A3 (de) | 2002-12-05 |
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