Classifications

• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
  • G01N2430/40—Dioxins

Definitions

• the present invention is directed to the detection of transformed Ah receptor and the consequent indirect detection of dioxin-like compounds by detection of transformed Ah receptor.
• dioxins have become the subject of intense scrutiny. This is due to their great toxicity and their assumed widespread presence in the environment .
• the toxicology of dioxins has been addressed principally through studies of their biological action using animal models and cell culture systems.
• the potential threat that dioxins present to human health has been addressed in only a limited manner through epidemiologic studies of populations known to have been exposed to dioxins.
• the environmental issues have been addressed through the study of the production, release, and degradability of dioxin.
• dioxins have been extensively studied in these ways, their exact mechanism of toxicity in biological systems and their extent of environmental distribution are unknown. This is due in part to the lack of a simplified method of assessing the exposure of biological systems to dioxins and related compounds.
• the present invention is directed to overcoming this deficiency in the art .
• TCDD 2, 3 , 7, 8-tetrachlorodibenzo-p-dioxin
• PCDD polychlorinated dibenzofurans
• PCB polychlorinated biphenyl family
• TCDD came to scientific and public attention in the early 1970s as a contaminant of the defoliants 2,4, 5-trichlorophenoxyacetic acid and 2,4- dichlorophenoxyacetic acid, notably through forest spraying programs in the U.S. and in Viet Nam.
• bioassays have been used extensively in the literature, they do not have practicable commercial utility.
• the chief disadvantage of such assays is their need for live cells or animals, making them unsuitable for a commercial test kit format.
• the enzymatic detection of P450IA1 is not very sensitive and requires sophisticated instrumentation.
• U.S. Patent No. 4,904,595 to Gierthy relates to an epithelial cell line and its use in an in vitro bioassay for dioxin-like activity. Upon exposure to dioxin, a morphological change is induced in the subject XBF cell line, cocultured with lethally irradiated 3T3 cells, to a flat cobblestone appearance, as compared with the fusiform high density state in control cultures not treated with dioxin.
• U.S. Patent No. 4,798,807 to Vanderlaan, et al discloses monoclonal antibodies which react with dioxin-like compounds and a method of using these antibodies in a sensitive immunoassay for such compounds. These antibodies recognize and bind to dioxin-like compounds in a competitive immunoassay.
• U.S. Patent No. 4,238,472 to Albro, et al discloses a radioimmunoassay method to detect dioxin-like compounds in environmental samples. This method involves combining a sample containing dioxin (emulsified with detergent) with a first antibody which binds to dioxin and radioactive I 125 labelled dioxin to form an antibody-I 15 -dioxin complex. This causes the labelled and unlabelled dioxin to compete for binding with the antibody. The complex is then reacted with a second antibody to form a precipitate containing dioxin.
• the present invention relates to a method of detecting, in a test sample, polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof which exhibit biological activity characteristic of such compounds via transformation of the Ah receptor.
• This method utilizes a heteromer formed from a plurality of proteins, one of which is an Ah receptor in inactive form.
• a test sample is brought in contact with the heteromer under conditions effective to bind any polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls and the like in the test sample to the Ah receptor.
• the binding of such ligands to the Ah receptor causes a complex containing Ah receptor bound to the ligand to dissociate from the heteromer and transform into an active state.
• the presence of the complex containing transformed Ah receptor bound to the ligand is then detected.
• the method of the present invention can be carried out with any one of several immunoassay test kit formats.
• One suitable format is a solid phase capture immunoassay test kit which includes the heteromer, an antibody with a region capable of binding to the complex and a label to permit detection of the antibody, and a solid support.
• a competitive immunoassay test kit format which includes the heteromer, a binding substance having a first region capable of binding to a solid support and a second region capable of binding to the complex, an analogue with a region capable of binding to the binding substance and a label to permit detection of the analogue, and a solid support.
• Another suitable format is a sandwich immunoassay test kit which includes the heteromer, a binding substance having a first region capable of binding to a solid support and a second region capable of binding to the complex, an antibody with a region capable of binding to the complex with a label to permit detection, and a solid support.
• the assay can be used directly in the field and does not contain any environmental contaminants other than what may be present in the sample to be measured. Samples can be assayed in the same day, and the results of many air, water, or soil samplings can be reported on-site. Such availability of on-site results allows for immediate evaluation of remediation efforts.
• Figure 1 shows a format for a modified solid phase capture immunoassay test kit.
• Figure 2 is a Western Blot gel showing the recognition of mouse liver cytosol proteins by anti-MAPS Antiserum 321. Lanes 5-11 and 13 depict antiserum recognition of 100 kDalton non-ligated form of the Ah receptor at various dilutions of antiserum.
• Figure 3 is a Western Blot gel showing the recognition of mouse liver cytosol proteins by anti-MAPS Antiserum 377. Lanes 1, 2 and 19 are protein standards stained for protein, Lanes 5 and 18 are cytosol stained for protein, and Lanes 12 through 15 depict antisera recognition of 100 kDalton non-ligated form of the receptor.
• Figure 4 is a Western Blot gel demonstrating the specific immuno-detection of Ah receptor transformed by TCDD.
• Figure 5A shows a nitrocellulose sheet dot blot demonstrating the capture and detection of TCDD-transformed Ah receptor onto nitrocellulose.
• Figure 5B is a graph of density versus volume of TCDD derived from scanning the dot blot of Figure 5A.
• Figure 6 shows a graph of optical density versus dose of TCDD.
• the present invention relates to a method of detecting, in a test sample, polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and structural analogues thereof which exhibit biological activity characteristic of such compounds.
• This method utilizes a heteromer formed from a plurality of proteins, one of which is an Ah receptor in inactive form.
• a test sample is then brought in contact with the heteromer under conditions effective to bind polychlorinated dibenzodioxins, polychlorinated dibenzofurans, polychlorinated biphenyls, and the like, in the test sample, to the Ah receptor.
• the binding of such ligands to the Ah receptor causes a complex containing active Ah receptor bound to the ligand to dissociate from the heteromer.
• the presence of the complex containing active Ah receptor bound to the ligand is then detected.
• the process by which the dioxin-like compounds bind to the Ah receptor and cause the active Ah receptor bound to the ligand to dissociate is known as transformation.
• the inactive Ah receptor exists as one of at least three proteins which form a cytosolic high molecular weight heteromer. Although the precise composition and structure of the heteromer is unknown, it is believed that heat shock protein 90 is another one of the constituent proteins.
• the Ah receptor Upon ligand binding to the Ah receptor, the Ah receptor dissociates from the complex and undergoes a conformational change to a heterodimer complex that has increased affinity for cationic exchangers and double stranded DNA. This process of activating the Ah receptor is essentially irreversible.
• activated Ah receptor bound to a ligand enters the nucleus and may bind to the nuclear regulatory sequence of several genes.
• DRE dioxin responsive element
• interactions between it and the activated Ah receptor are believed to lead to enhanced gene expression.
• the Ah receptor with dioxin-like compound ligands are not themselves toxins, the enhanced gene expression caused by its binding to the dioxin responsive element is believed to be the basis of the toxic response to dioxin-like compounds.
• the transformation phenomena is discussed in more detail in G. P. Landers et al. , "Review Article -- The Ah Receptor and the Mechanism of Dioxin Toxicity," Biochem. J. , vol. 26, pp. 273-87 (1991) and S.
• PCBs Polychlorinated Biphenyls
• PCDDs Dibenzo-Dioxins
• PCDFs Dibenzofurans
• TEZs Toxic Equivalency Factors
• the heteromer containing the Ah receptor can be obtained from any number of sources. Although the heteromer has been identified in several human tissues and cells in culture, including lung, liver, kidney, placenta, B lymphocytes, and thymus, it is preferably obtained from other mammals for ease of availability.
• the heteromer is present in rodent liver, thymus, lung, kidney, brain, testis, and skeletal muscle cells.
• a particularly preferred source of the heteromer is a cytosol fraction of mammalian hepatocytes.
• the heteromer can be obtained by isolating liver cytosol from male Hartley guinea pigs, according to E. C. Henry, et al . , "Characterization of Multiple Forms of the Ah receptor: Comparison of Species and Tissues," Biochem, 28:6430-40 (1989) , and frozen or lyophilized in five milliliter aliquots contained in glass or plastic test tubes.
• the process of the present invention is useful for detecting dioxin-like compounds in a variety of test samples.
• test samples can be an environmental matrix of air, water, or soil.
• the assay can be used to detect dioxin-like compounds in the body fluids (e.g., blood) of humans or animals.
• the present detection method is desirably carried out in any conventional test kit format.
• the immunoassay can be a solid capture, competitive, or sandwich immunoassay.
• the solid phase capture immunoassay test kit includes the heteromer, an antibody capable of binding to the complex and having a label to permit detection, and a solid support.
• the solid support can either be sold as part of the test kit or separate from it.
• the heteromer is contacted with the test sample.
• the resulting mixture is contacted with the solid support so that the complex binds to the support .
• the antibody is contacted with the bound, active Ah receptor.
• the label on the antibody can then be detected.
• the mixture of heteromer and test sample can be contacted with an affinity matrix so that the complex binds to the affinity matrix.
• the complex After removal of unbound material, the complex is eluted from the affinity matrix and allowed to contact and adsorb to the solid support. The labelled antibody is then contacted with the adsorbed complex to permit detection.
• the affinity matrix can also be used to bind to the complex and thereby separate the complex from the remainder of the test sample-heteromer mixture in the sandwich and competitive formats. In each, the complex can be subsequently eluted from the affinity matrix and into absorptive contact with the solid support.
• the competitive immunoassay kit includes the heteromer, a binding substance having a first region capable of binding to a solid support and a second region capable of binding to the complex, an analogue with a region capable of binding to the binding substance in the second region and having a label to permit detection of the analogue, and a solid support.
• this competitive immunoassay test kit format is utilized in the detection method of the present invention, the binding substance is contacted with the solid support so that the former binds to the latter.
• the analogue is then contacted with the binding substance under conditions causing the analogue to compete with the complex, formed during contact between the test sample and the heteromer, to be bound to the second region of the binding substance. After such competition, the step of detecting takes place.
• the sandwich immunoassay kit contains a binding substance having a first region capable of binding to a solid support and a second region capable of binding to the complex, the heteromer, an antibody with a region capable of binding to the complex containing active Ah receptor bound to the ligand and having a label to permit detection of the antibody, and a solid support.
• the binding substance is contacted with the solid support.
• the test sample is contacted with the heteromer
• the mixture of test sample and heteromer is placed in contact with the solid support.
• the complex binds to the second region of the binding substance.
• the labelled antibody is contacted with the complex bound to the solid support.
• the label on the antibody can be detected.
• the solid support used in any of these immunoassay test kit formats may be any water insoluble, water suspendible solid material conventionally utilized in such kits. Suitable examples are polymeric membranes, plastic or glass beads, test tubes, or microtiter plates.
• the binding substance in the complex, containing active Ah receptor bound to the ligand may be bound to the solid carrier by covalent binding or adsorption. When test tubes or microtiter plates are utilized, such bonding takes place at the inner walls of these carriers.
• the kit can be merchandised with the binding substance already bound to the solid support.
• Such application to the solid support surface is achieved by contacting the binding substance with the solid support and maintaining such contact for sufficient time to permit the first region of the binding substance to bond to the solid support. Typically, such contact takes one to eighteen hours, preferably four hours.
• the non-adhered binding substance is then separated from the insolublized binding substance (i.e., that which is bound to the solid support) and the solid support is then washed.
• test sample and the heteromer are placed in contact with each other and allowed to incubate for sufficient time to permit transformation. Typically, such transformation takes two hours. Such contact desirably is followed by contacting the test sample and heteromer mixture with a solid support.
• the complex binds directly to the solid support, while the complex binds indirectly (i.e., through the binding substance) to the solid support in the competitive assay or sandwich immunoassays .
• residual test sample and heteromer mixture is separated from the insolublized material bound to the solid support. The insoluble material is then washed.
• the labelled antibody for the solid phase capture or the labelled analogue for the competitive immunoassay test kit are contacted with insolubilized material bound to the solid support, such contact is maintained for sufficient incubation time so that the labelled material bonds indirectly to the solid support. Typically, one hour to eighteen hours, preferably two hours, is sufficient for such binding.
• the unbound material is then separated from the insolubilized material, and the insolubilized material is then washed.
• a labelled analogue or antibody (as the case may be) is needed to detect the extent to which that analogue or antibody indirectly bonds to the solid support.
• detection preferably involves a quantitative measurement of the labelled material.
• the labeled antibody bonds directly to the complex so that the detection procedures directly determine the amount of complex formed.
• the labelled analogue which is actually detected is insolubilized at sites where the complex is not present. Thus, the amount of complex formed must be determined indirectly from the amount of analogue detected.
• the label can be a colored, fluorescent, chemiluminescent, radioactive, or enzymatic material conjugated to the antibody or analogue, or a colored, fluorescent, chemiluminescent, radioactive, or enzymatic material conjugated to a secondary binding substance such as an antibody that binds to the binding substance that interacts with the complex.
• an antibody capable of binding to the complex containing active Ah receptor bound to a dioxin-like compound ligand is used.
• the antibodies can be in polyclonal or monoclonal form.
• a particularly preferred procedure for obtaining detection antibodies is to synthesize the following peptide sequence modified from the mouse Ah receptor N-terminus :
• the unpurified peptide is coupled to ovalbumin using maleimidobensoyl-N- hydroxysuccinimide ester with a yield of 9 to 22 moles of peptide per mole of ovalbumin.
• Rabbits are then immunized with 0.5 to 1 mg quantities of the antigen at bi-weekly intervals and bled after 8 weeks and thereafter. Serum is obtained from the blood of the rabbits.
• the peptide SEQ. ID No. 1
• the peptide (SEQ. ID No. 1) is covalently linked to an iodoacetamide derivatized column through the sulfhydryl group on the terminal cysteine of the peptide.
• Antibodies to the Ah receptor N-terminus are purified from the serum by affinity chromatography on the above-described column.
• the preferred label for the antibody is commercially available anti-rabbit IgG alkaline phosphatase conjugate.
• a labelled analogue which is capable of binding to the second part of the binding substance.
• Suitable analogues include the above peptide sequence (i.e. SEQ. ID. No. 1) coupled to alkaline phosphatase.
• the analogue can be an antigen which has the same immunological properties and is immunologically equivalent to the activated Ah receptor. See U.S. Patent No. Re 32, 696 to Schuurs et al . which is hereby incorporated by reference.
• the preferred procedure for synthesizing the labeled analogue is to couple alkaline phosphatase to the peptide sequence (SEQ. ID. No.
• Enzymatic labels are well known in the art. Examples of such labels include alkaline phosphatase, horseradish peroxidase, glucose-6-phosphate, 3-galactosidase, xanthine oxidase, catalase, urease, glucose oxidase, galactose oxidase, ⁇ -glucuronidase, and ⁇ -B-glucosidase.
• Such labels are detected by the conversion of substrates to measurable product colorimetrically, fluorometrically, and spectrophotometrically using devices well known in the art. These instruments generate an optical density value which can be converted to a dioxin-like compound concentration by comparison to a standard curve.
• the antibody or analogue can be directly labeled.
• Suitable colored labels include fluorescent, chemiluminescent and colorimetric. These labels are detected by spectrophotometry or densitometry. These instruments generate an optical density value which can be converted to a dioxin-like compound concentration by comparison to a standard curve.
• Suitable fluorescent labels for the antibody or analogue include fluorescein, rhodamine and their derivatives. These labels are detected by fluorimetry. These instruments generate an optical density value which can be converted to a dioxin-like compound concentration by comparison to a standard curve.
• Suitable chemiluminescent labels include luminol, isoluminol, acridinium esters, thioesters, sulfonamides, and phenathridinium esters. These labels generate an optical density value which can be converted to a dioxin-like compound concentration by comparison to a standard curve. Such labelling systems produce a long-lived glow of light. This glow can be detected with luminometers, photomultiplier tubes, and solid state detectors.
• the competitive and sandwich immunoassays both utilize a binding substance with a first region capable of binding to the solid support and a second region capable of binding to a complex containing active Ah receptor bound to a dioxin-like compound ligand.
• the binding substance is preferably selected from the group consisting of an antibody, a dioxin responsive element, and a portion of the dioxin responsive element.
• binding substance is in the form of an antibody
• antibodies can be polyclonal or monoclonal.
• the binding substance can be composed of calf thymus DNA adsorbed or covalently coupled to a solid support or specific DNA sequences adsorbed or covalently coupled to a solid support.
• the DNA sequences for the dioxin responsive element of cells in various species has been the subject of extensive investigation.
• T. A. Gasiewicz "Accelerated Communication -- alpha-naphthoflavone Acts as an Antagonist of 2,3,7, 8-Tetrachlorodibenzo-p-Dioxin by Forming an Inactive Complex with the Ah Receptor, "Molecular Pharmacology. " 40:607-12 (1991) utilizes the following nucleotide sequences:
• Both of these sequences are prepared with a DNA synthesizer with the latter being synthesized with a 5 prime amino modifier C6-TFA.
• This linker provides a primary amine with the 6-carbon spacer at the 5' end.
• these 2 nucleotide sequences are dissolved in 20 mM (3- [N-Morpholino]propanesulfonic acid buffer pH 7.6 plus 1 mM ethylenediaminetetraacetic acid in equimolar amounts, hybridized by heating to 90°C for five minutes, and cooled to 37°C overnight. The hybridized dioxin-responsive elements are then covalently coupled to cyanogen-bromide activated sepharose beads.
• the apparatus of Figure 1 is utilized.
• This device includes holder 2 having a plurality of small polypropylene columns 4. Columns 4 have open ends and are each fitted with a affinity matrix 6 at the bottom thereof.
• Solid phase capture unit 8 includes capture member holder 10 formed from e.g., a polyethylene 96-well flat bottom plate 12 with holes in the bottoms of each well. Nitrocellulose discs constituting capture membranes 14 are cemented into these holes.
• Container 16 with vacuum manifold outlet 18 is designed to fit under capture membrane holder 10.
• cytosol with heteromer containing inactive Ah receptor is mixed with a test sample that may contain dioxin- like compounds. After the mixture incubates for a few hours, it is pipetted into each of columns 4 in holder 2. Effluent passing through the columns drains into a waste container (not shown) .
• affinity matrix 6 can be utilized in the sandwich and competitive immunoassay formats of the present invention.
• the affinity matrix is contacted with the mixture of test sample and heteromer so that the complex binds to the matrix.
• the bound complex is then eluted from the matrix and brought in contact with and absorbed on a solid support.
• the immunoassays of the present invention have a number of potential uses.
• One use for this assay would be the one step determination of toxic equivalent factors ("TEFs") .
• TEFs are a measure of the toxic potential of Ah receptor-dependant toxins and can be used in the hazard and risk assessment of such compounds.
• the assay could be used to screen for anti-estrogenic drugs used for mammary tumor therapy.
• the assay could also be used to screen potential natural or synthetic TCDD antagonists which may have potential as anti-promotional agents or in cancer prevention. It could be a rapid screen for dioxin-like toxicity in pharmaceutical and agrichemical products.
• the assay could be used as an endpoint in experiments for studying the cellular events that effect Ah receptor transformation in human cell lines, in order to understand human susceptibility to dioxin-like compounds.
• the assay could be used to determine exposure status to TCDD and dioxin-like compounds in human or animal tissues and cells, the response of human Ah receptor to TCDD and PCBs, and the levels of Ah receptor in malignant cells.
• MAPS Multiple antigen peptide synthesis
• mice were sacrificed and the livers perfused with 1.15% KCl in MENG buffer.
• Hepatic cytosol fractions were prepared by homogenizing the liver in 3 times the volume of MENG buffer (Poland et al . , Molecular Pharmacology, vol. 39, 20-26, (1990), which is hereby incorporated by reference) .
• the supernatant (S-9) was centrifuged at 100,000 x g for 60 minutes and the resulting cytosol fraction was carefully removed and quickly frozen for storage at -80°C.
• lanes 1-3 and 20-22 represent protein standards stained for protein. Standards were (from top to bottom) 116, 97.4, 66, and 45 kDaltons. Lanes 4 and 19 were pre-stained protein standards; these were 205, 116.5, 80, and 49.5 kDaltons, top to bottom, respectively. The total cytosol stained for protein appears in lanes 5 and 18. All other lanes were immunostained with antisera.
• Anti-MAPS 321-1 (1:11) in lanes 6 and 7, anti-heat shock protein 90 (HSP90, Source) (1:500) in lane 8, anti-MAPS 321- 2 (1:11) in lanes 10 and 11, anti-MAPS 321-2 (1:50) in lanes 12 and 13, anti-MAPS 321-2 (1:250) in lanes 14 and 15, and anti-MAPS 321-2 (1:1000) in lanes 16 and 17.
• HSP90, Source anti-heat shock protein 90
• anti-MAPS 321- 2 (1:11) in lanes 10 and 11
• anti-MAPS 321-2 (1:50) in lanes 12 and 13
• anti-MAPS 321-2 (1:250) in lanes 14 and 15
• anti-MAPS 321-2 (1:1000) in lanes 16 and 17.
• the results indicate that the second bleeding rabbit 321 was producing antibodies capable of recognizing the 100 kDalton, nontransformed state of the Ah receptor at a titer of 1:50.
• Lanes 1, 2 and 19 of Figure 3 represent protein
• Molecular weights represented were 116, 97.4, 66, and 45 kDaltons.
• the pre ⁇ stained standards in lanes 3 and 18 represent molecular weights of 205, 116.5, 80, and 49.5 kDaltons. Cytosol stained for protein was in lanes 4 and 17. All other lanes were immunostained with antisera. The following antisera (with dilution) were used: Anti-MAPS 377-2 (1:250) in lanes 6 and 8, anti-MAPS 377-2 (1:50) in lanes 9 and 10, anti-MAPS 377-2 (1:11) in lanes 11 and 16, anti-MAPS 377-1 (1:11) in lane 13, and anti-HSP90 (1:500) in lanes 14 and 15.
• Anti-043 polyclonal antiserum was prepared by immunizing rabbits with sequence ID No. 1 coupled to ovalbumin via maleimidobensoyl-N-hydroxysuccinimide ester coupling with a yield of 9 to 22 moles of peptide to carrier. Rabbits were immunized with 0.5 to 1 mg quantities of the antigen at bi-weekly intervals and bled after 8 weeks and thereafter. Serum was obtained from the blood of the rabbits and purified on an affinity column made from peptide sequence ID No. 1 linked to iodoacetamide derivatized agarose beads.
• Dioxin-responsive elements were made by hybridizing oligonucleotide sequences ID No. 6 (166 nmoles) and ID No. 7 with sequence 7 containing an N-TFA-C6 linker on the 5'end (97 nmoles) . These oligonucleotide were dissolved in 20 mM (3- [N-morpholino] propanesulfonic acid buffer pH 7.6 plus 1 mM ethylenediaminetetraacetic acid and hybridized by heating to 90°C for five minutes, and cooled to 37°C overnight. The hybridized dioxin-responsive elements were then covalently coupled to cyanogen-bromide activated sepharose beads as follows.
• Dry beads (1.14 g) were placed in a scintered glass funnel attached to a vacuum aspirator. The beads were swollen by washing sequentially with ice cold 250 ml 1 mM HCl, cold 300 ml deionized water, and cold 40 ml 10 mM potassium phosphate buffer pH 8.0. The beads plus DREs were then incubated 16 hours at room temperature with gentle rocking. The beads were then washed with 200 ml water and 100 ml 1 M ethanolamine pH 8, resuspended in 5.6 ml ethanolamine buffer, and incubated 6 hours at room temperature with rocking.
• the beads were then washed with 100 ml water, 100 ml 10 mM potassium phosphate buffer pH 8.0, 100 ml 1 M potassium phosphate pH 8.0, 100 ml 1 M KCl, and 100 ml 10 mM Tris (hydroxymethyl) aminomethane-HCl (pH 7.6) containing 0.3 M NaCl, 1 mM ethylenediaminetetraacetic acid, and 0.02 % sodium azide.
• the resulting affinity matrix was made into two 2.4 ml bed volume open columns in 10 ml syringe barrels and stored at 4°C.
• HEDG buffer 25 mM N- [2-hydroxyethyl]piperazine-N' - [2-ethanesulfonic acid] , 1.5 mM ethylenediaminetetraacetic acid, 1.0 mM dithiothreitol, pH 7.6
• the livers were homogenized, and the extract centrifuged at 12,500 x g for 20 minutes. The supernatant was then re-centrifuged at 100,000 x g for 60 min, and the supernatant (hepatic cytosol) was frozen at -80 C.
• the filtered cytosols were then passed through an affinity matrix column, followed by 2 x 4ml HEDG, 2 x 4 ml HEDG plus 350 mM NaCl, and 2 x 4 ml HEDG plus 600 mM NaCl. The last washes were collected and 800 ⁇ g bovine serum albumin in 800 ⁇ l water was added to each followed by 8.8 ml 20% ice cold trichloroacetic acid. The proteins were allowed to precipitate on ice for 30 min and centrifuged in a clinical centrifuge for 30 min.
• the supernatant was discarded and the precipitate was dissolved with 100 ⁇ l 1 M TRIS pH 8.0, 400 ⁇ l water and 500 ⁇ l 2X sample buffer (5 mM Tris, 0.05 % bromphenol blue, 2 % mercaptoethanol, 2 % sodium dodecyl sulfate, 10 % glycerol, pH 6.8) .
• the dissolved proteins were denatured by boiling 5 min and 100 ⁇ l each analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Laemmli, 1970) followed by blotting onto polyvinylidene difluoride according to Towbin et al. (1979) . Protein standards were run in adjacent lanes.
• the lane containing protein standard was stained with 0.1 % amido black 10B in 7 % acetic acid and destained in 7 % acetic acid.
• the lanes containing the column eluants were incubated in blocker buffer (5 % non-fat dry milk in TBST (50 mM TRIS, 150 mM NaCl, 0.02 % Tween 20, pH 7.5)) . Incubation conditions were 50 ml and 1 hour. The blot was then probed with 40 ml anti-043 antiserum (0.1 % in blocker buffer) for one hour.
• the blot was then washed 3 times, five minutes each, in 100 ml TBST, followed by incubation with 40 ml 0.1 % anti-rabbit IgG/alkaline phosphatase conjugate in blocker buffer for one hour, followed by 2 five minute washes with 100 ml TBST, one five minute wash with
• alkaline phosphatase development buffer 5.5 g Trizma-base, 0.8 g Trizma-HCl, 2.9 g NaCl, 5.1 g MgCl 2 -6H 2 0
• the blot was developed with 3.3 mg 5-bromo-4-chloro-3-indolyl-phosphate and 6.6 mg nitroblue tetrazolium in 20 ml alkaline phosphatase development buffer for 5 min, washed several times with water, and dried.
• Lane A was loaded with affinity matrix eluant from solvent treated cytosol .
• Lane B was loaded with affinity matrix eluant from 10 nM TCDD treated cytosol.
• Lane C was loaded with molecular mass standards, identified by kD in vertical column.
• results demonstrate that the anti-043 antiserum detects a single protein band in the column eluant.
• the presence of the protein is TCDD dependent, since it appears in the TCDD treated sample but not the solvent treated sample.
• the estimated molecular mass (circa 105 kD) is identical to guinea pig Ah receptor. It binds to a dioxin-responsive element as transformed Ah receptor is predicted to do. It is recognized by an antiserum made to a peptide sequence specific and conserved among mammalian Ah receptors.
• these results demonstrate that the treatment of cytosol with dioxin-like compounds followed by isolation and immunodetection of transformed Ah receptor can detect the presence of dioxin- like compounds.
• the immunodetection of a single TCDD-responsive band indicates the protocol is specific to the detection of transformed Ah receptor and can be modified into a simple immunoassay test kit without the need of resolving unwanted immuno-reactive proteins.
• FIG 5 volumes given are equivalent to original cytosol preparation.
• (-) solvent treated cytosol
• (+) 10 nM TCDD treated cytosol.
• Figure 5A shows the nitrocellulose sheet with color due to the presence of transformed Ah receptor.
• Figure 5B are averaged density data derived from scanning the dot blot in Figure 5A. Hatched bars: Sovent control. Solid bars: 10 nM TCDD treatment. Results indicate that nitrocellulose efficiently captures the transformed Ah receptor, with low background. Increasing volumes of TCDD treated sample produced increasing color development which could be seen by the naked eye, while increasing volumes of solvent treated sample showed no or little color development. Good sensitivity and linear capture was seen in the range of 50 to 200 ⁇ l sample loadings.
• Example 6 This example is set forth with reference to Figure 6. The methodology was identical to Example 2 except for the following changes.
• Affinity matrix columns were prepared as described in Example 2 but were formed into multiple 100 ⁇ l bed volume columns in 1 ml tuberculin syringe barrels. Cytosol (500 ⁇ l) was transformed with variable amounts (0, 0.016, 0.08, 0.4, 2, 10 nM; corresponding to 0, 13, 64, 320, 1600 pg TCDD respectively) . After loading the samples onto the mini-columns, they were washed with 2 x 160 ⁇ l HEDG, 2 x 160 ⁇ l HEDG plus 350 mM NaCl, and 2 x 160 ⁇ l HEDG plus 600 mM NaCl.
• Example 5 Methodology was identical to Example 3 except that the volume of cytosol treated per sample was increased from 500 ⁇ l to 800 ⁇ l, and the dose range of TCDD used was 0, 0.0032, 0.016, 0.08, 0.4 and 2 nM.
• the resulting data showing optical density at different TCDD concentrations is set forth below in Table 1. Table 1
• Results show that, similar to Example 3, the response is essentially saturated between 0.08 and 2 nM. However, increasing the amount of cytosol allowed the detection of 0.016 nM or 5 pg TCDD.
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)

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