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WO1995006257A1 - Dosage par transfert de marqueur induit par recepteur - Google Patents

Dosage par transfert de marqueur induit par recepteur Download PDF

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Publication number
WO1995006257A1
WO1995006257A1 PCT/US1994/009553 US9409553W WO9506257A1 WO 1995006257 A1 WO1995006257 A1 WO 1995006257A1 US 9409553 W US9409553 W US 9409553W WO 9506257 A1 WO9506257 A1 WO 9506257A1
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WIPO (PCT)
Prior art keywords
tnf
receptor
iigand
complex
detecting
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PCT/US1994/009553
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English (en)
Inventor
Bruce A. Beutler
Alexander Poltorak
Karsten Peppel
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Board Of Regents, The University Of Texas System
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Publication date
Application filed by Board Of Regents, The University Of Texas System filed Critical Board Of Regents, The University Of Texas System
Priority to AU76031/94A priority Critical patent/AU7603194A/en
Publication of WO1995006257A1 publication Critical patent/WO1995006257A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]

Definitions

  • the present invention relates generally to the field of molecular biology and to its application in detection of hormones and hormone receptors.
  • the invention concerns methods of detecting hormones or hormone receptors at subfemtomolar concentrations.
  • the disclosed methods have particular utility in detecting tumor necrosis factor (TNF) and TNF receptor which are thought to be associated with the presence and progress of infectious and neoplastic diseases.
  • TNF tumor necrosis factor
  • Radioimmunoassay a technique in which antibodies that react specifically with a given macromolecule are added to the assay sample, together with a labelled "tracer" (for example, radioiodinated hormone) . Competition for the binding of the tracer is taken to indicate the presence of the macromolecule within the sample. Radioimmunoassay is limited in sensitivity by the affinity of the antibody. Typically, the detection limit is in the picomolar range.
  • Subpicomolar detection of macromolecules may sometimes be afforded by ELISA assay techniques, employing an excess of a binding reagent (usually also an antibody) as a solid phase Iigand to capture the target macromolecule.
  • a binding reagent usually also an antibody
  • the macromolecule is then detected by a second antibody which binds to a distinct epitope on the macromolecular surface.
  • Final detection of the macromolecule is achieved indirectly, through the use of an enzyme-linked tertiary antibody.
  • the sensitivity of the ELISA assay is limited by the affinity of the reagents applied at each step.
  • Other problems with this method include a relatively high signal-to-noise ratio imposed by the washing required to remove unbound reagents from the system.
  • ELISAs may suffer from problems related to diffusion of the macromolecule, which may escape immobilization in the first step of the reaction.
  • cytokines exert biological effects at concentrations beneath those that are detectable through the use of conventional assays (e . g. , biological assay, radioimmunoassay, or ELISA assay) .
  • conventional assays e . g. , biological assay, radioimmunoassay, or ELISA assay
  • tumor necrosis factor may be produced in many forms of human cancer, as well as in diverse infectious and inflammatory disorders, and is thought to have significant clinical effects.
  • interleukin-1, interleukin-2, and many other protein hormones globally referred to as cytokines Cytokine receptors shed from the cell surface may exert biological effects by preventing the cytokines that interact with them from binding the cell-surface receptors.
  • TNF tumor necrosis factor
  • the RIA for TNF is limited by antibody affinity, and cannot normally detect subpicomolar concentrations of the protein in plasma.
  • the ELISA methods which effectively concentrate TNF out of solution through the use of an immobilized antibody and then detect TNF through the use of a second antibody, are to some degree beholden to the specificity and to the off-rate of the detecting reagent. Practically speaking, the method is limited by the volume of plasma that may be applied to the ELISA plate. The positive signal elicited upon assay of normal plasma or serum has led to the assumption that TNF exists in picomolar concentration in the biological fluids of healthy individuals.
  • the present invention seeks to overcome these and other drawbacks inherent in the prior art by providing highly sensitive methods of detecting either component of Iigand:receptor complexes.
  • the methods of the present invention provide a means of detecting the active form of polypeptide hormones in human plasma with greater sensitivity than any currently available method.
  • the new methods have been applied particularly to the detection of tumor necrosis factor (TNF) , a polypeptide hormone produced by macrophages and other cells in vivo; however, broader application is envisioned.
  • TNF tumor necrosis factor
  • polypeptide hormones should be detectable with adaptation to measurement of soluble receptors for hormones, and to the measurement of hormones and receptors in plasma and other human biological fluids (e.g., cerebrospinal fluid, urine, amniotic fluid, etc.)
  • the general basis for detecting subpicogram levels of polypeptide macromolecules, either ligands or receptors, is the initial formation of a relatively tight ligand/receptor complex.
  • the complex is then "captured” by reacting with a cross-linking agent to form a covalent bond between the Iigand and receptor.
  • the complex may be detected by suitably sensitive methods; generally by measuring or visualizing radiolabeled components of the complex. While the radiolabelled complex has been identified by autoradiography after physical separation from the unreacted Iigand employing gel electrophoresis, immobilization of the complex on a solid support coated with antibody against TNF may allow removal of the unreacted Iigand by a washing procedure so that an electrophoretic step is unnecessary. Other methods of separation may include the use of antibody coated magnetic beads, size separation methods such as sieving, ion exchange chromatography and electrofocusing.
  • Cell surface receptors are molecules that have evolved so as to engage a specific hormone with high affinity, usually leading directly to transduction of a molecular signal specified by the hormone:receptor complex. Receptors usually have evolved to bind to the hormone that targets them with high affinity as a result of a complimentary tertiary structure, and extensive surface contact occurring between the two molecular species. Receptors have evolved so as to engage no components of the milieu interior other than the hormone itself. Many receptors are "shed" from the cell surface as a result of proteolysis, and continue to circulate for some time in an active form, engaging hormones in the extracellular compartment. At times, a single hormone may become bound to more than one monomeric chain.
  • Tumor necrosis factor is a trimeric protein, which engages three receptor subunits at the cell surface, triggering a biological response.
  • bivalent, or in some instances, trivalent, forms of a receptor may yield a far stronger interaction with a given hormone species, since two or three binding sites are involved rather than single binding site.
  • the affinity constant of binding is to some extent multiplied by the presence of more than one interaction.
  • a bivalent form of TNF receptor has been described which binds to TNF with far greater affinity than a monomeric form of the receptor (Peppel et al . , 1991)
  • the macromolecular Iigand should form a stable complex with the receptor molecule with a binding affinity above 10 9 K a .
  • Monoclonal antibodies typically exhibit affinities in the range of 10 5 -10 6 K a and would therefore need to be employed in impractically large quantities for the methods of the present invention.
  • the present invention requires addition of TNF binding protein at a saturating concentration (approximately 0.2 ⁇ g of binding protein per milliliter of plasma) .
  • a typical antibody would need to be added at 1000 times this concentration to achieve a successful measurement (and correspondingly, 1000 times as much radionuclide would need to be used) .
  • the sensitivity of many assays based on the use of antibodies is limited by the affinity of the antibody:antigen interaction. This is particularly true for competition based assays where a radiolabelled tracer is expected to compete with unlabeled target molecule present in a biological solution.
  • the method of the present invention is not a competitive assay limited by the affinity of the binding Iigand. For example, with a low affinity binding protein, larger amounts of labeled binding protein must be added to the system in order to measure the target molecule.
  • Soluble receptors suited for practice of the invention may be isolated from natural sources or produced by recombinant means (Peppel, et al . , 1991).
  • Truncated receptor molecules may be useful, for example, those lacking transmembrane or cytoplasmic domains that retain capacity to interact with the appropriate ligands. While in some cases, truncated forms of receptors may be highly unstable in vivo, engineered chimeric proteins will in many cases become the preferred forms of the receptor.
  • Preferred partners in the chimeric molecules include IgG, heavy chain immunoglobulins. Particularly preferred embodiments include the CH 2 through CH 3 domains of mouse IgGl or alternatively, of the human IgG heavy chain.
  • a chemically modified or mutant form of binding protein may be employed to facilitate detection after complex formation.
  • a biotinylated complex may be measured using an avidan/peroxidase detection system.
  • Certain modifications of the IgG heavy chain, such as the incorporation of a metallothionein domain into the sequence in order to complex radiolabelled copper may increase the sensitivity of detection.
  • Cross-linking the polypeptide macromolecule with the receptor assures that there will be no dissociation of the complex.
  • Covalent bonds may be formed with a variety of chemical reagents including disuccinimidyl suberate (DSS) , DTT, formaldehyde, glutaraldehyde and paraformaldehyde.
  • DSS disuccinimidyl suberate
  • Cross-linking reagents such as DSS form covalent cross-links relatively rapidly so that treatment of the Iigand receptor complex usually requires approximately 30 minutes. Separation of the receptor Iigand complex from unreacted Iigand may be performed using a variety of methods well known to those of skill in the art.
  • the complexes are immunoprecipitated and then subjected to electrophoresis under denaturing conditions in a polyacrylamide gel.
  • the complex differs in electrophoretic properties from the mobility of the soluble receptor, and therefore the presence of the polypeptide macromolecule is detectable by observing a shift band with slower mobility than the receptor itself.
  • Electrophoresis is a convenient and rapid method of detecting which has provided sensitivity in the range of 10 femtomolar when radiolabelling with 125 ⁇ is employed.
  • Sensitivity of detection will, of course, depend to some extent on the ability to detect small amount of complex. This may be accomplished through labeling of the Iigand or polypeptide macromolecule. Suitable labels include 32 P, 125 I, 99 Tc and 67 Cu. A preferred label is the 131 ⁇ label, which permits detection into the attomolar range. In view of its shorter half-life and its emission of a beta particle that is readily detectable on film with the use of fluorogenic reagents, 131 I for example is expected to afford even higher assay sensitivity.
  • chimeric fusion partner for the soluble receptor.
  • the fusion partner may be selected for its binding properties, for example, permitting easy separation from reaction mixtures.
  • Chimeric fusion partners such as IgG heavy chain are readily separated by affinity chromatography using an anti IgG column.
  • Numerous other fusion partners, which may be employed in engineering the primary protein include galactose-specific lectin, immunoglobulin binding protein, maltose binding protein, carbohydrate recognition region of galactose specific lectin, polyhistidine and glutathione transferase, to mention a few. Chimeric proteins engineered with these entities are readily separated from sample mixtures by the use of appropriate affinity columns.
  • the invention additionally encompasses a method for detecting a soluble hormone receptor.
  • This operates in the same way as the method for detecting the Iigand or polypeptide macromolecules that bind to a designated receptor.
  • one should be able to detect any receptor, particularly soluble receptors such as hormone, cytokine and epitopic domains of antigen receptors.
  • One will desire first to identify a Iigand that binds to a particular receptor. The binding should be selective. Natural receptors are suitable but synthetic receptors are also envisioned, including modified natural receptors so long as there is relatively high binding affinity and specificity.
  • the Iigand is preferably labeled, for example with a radio-label or fluorescent label.
  • Iigand receptor complex is accomplished by any one of the several methods previously described, preferably by electrophoresis.
  • the disclosed methods of detecting soluble receptors or their ligands are applicable to the determination of biomolecules in samples including plasma, urine, cerebrospinal fluid and amnionic fluid, etc.
  • the methods of detection are ideally suited for determining extremely low levels of biologically active species such as might exist in pathological conditions.
  • Compounds particularly amenable for detection in this manner include hormones and cytokines; for example, IL-l ⁇ , IL-1/3, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, TNF, lymphotoxin, GM-CSF, GCSF, MCSF, interferon- ⁇ , Fas Iigand, erythropoietin, growth hormone, insulin, TGF- ⁇ , TGF-j ⁇ , FGF, and so forth.
  • hormones and cytokines for example, IL-l ⁇ , IL-1/3, IL-1 receptor antagonist, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, TNF, lymphotoxin, GM-CSF, GCSF, MCSF, interferon- ⁇ , Fas Iigand, erythropoietin, growth hormone, insulin, TGF- ⁇ , TGF-j ⁇ , FGF, and so forth.
  • excess labeled recombinant TNF receptor extracellular domain is incubated with a sample suspected of containing TNF.
  • the labeled receptor is covalently cross-linked with the TNF by employing an appropriate cross-linking agent.
  • the complex is then immunoprecipitated and electrophoresed on polyacrylamide gel under denaturing conditions. Shifted bands, that is bands having different mobility from the soluble TNF recombinant receptor, are identified and quantitated. The position of shifted bands on the gel readily identifies monomeric and oligomeric TNF species covalently bound to the receptor. All TNF complexes have slower mobility than the recombinant TNF receptor.
  • a particularly preferred recombinant TNF receptor is the 55 kDa human TNF receptor domain covalently linked with a chimeric fusion partner.
  • a preferred fusion partner is mouse immunoglobulin IgG-* ⁇ heavy chain, or the human counterpart IgG heavy chain.
  • other recombinant TNF receptors and fusion partners are expected to be suitable, including treated versions of recombinant TNF receptor and mutant TNF receptor such as encoded by TNF DNA altered by chemical mutagenesis or site directed mutagenesis or site directed mutagenesis. This assay has capitalized upon advantages offered by the TNF binding protein. First, the specificity of the 55 kDa TNF receptor (or any receptor) for its natural Iigand is ensured by evolutionary processes.
  • TNF receptor a soluble form of the TNF receptor will engage very few molecules in plasma other than the TNF or lymphotoxin molecules themselves. This situation does not obtain when antibodies (whether polyclonal or monoclonal) are used as a reagent for the detection of TNF, as in an ELISA assay.
  • the bivalent receptor will engage only active molecules (i.e., molecules operationally defined as active based on their content of two receptor binding sites, which can act to aggregate the natural receptor on the cell surface) with high affinity. Denatured TNF, or TNF fragments, will not be detected by the assay described herein. Once bound to TNF in solution, the detecting reagent is readily crosslinked to form an irreversible adduct.
  • the assay reaches an extraordinary level of sensitivity when 131 ⁇ is employed as the radionuclide marker.
  • the use of an agent which yields such a strong signal is made feasible by virtue of the singularly low signal-to-noise ratio of the assay system.
  • TNF tumor necrosis factor
  • soluble receptors for which ligands have not yet been identified e . g. , the Fas antigen
  • a labelled version of a hormone may be used to detect soluble forms of the receptor; for example in measuring the expression of TNF binding protein in transgenic mice.
  • the assay is envisioned as applicable to the detection of a wide variety of proteins for which soluble receptors can be synthesized. As such, the label-transfer technique herein described is expected to be the assay of choice when extreme sensitivity and specificity are required.
  • Yet another aspect of the present invention is the determination of the presence of neoplastic or infectious diseases. This involves measuring the amount of TNF in accordance with the disclosed method. Mammals suspected of having such disease are expected to exhibit varying levels of plasma TNF. These levels may be indicative of the progress and course of the disease. Surprisingly, TNF has not been detected in normal individuals employing the new method. This suggests that even low TNF levels in plasma would suggest developing problems or an underlying disease state.
  • kits for use in identifying and quantifying TNF or TNF receptors are contemplated.
  • kits for use in connection with polyacrylamide gel-based separation techniques are contemplated.
  • Such kits will generally comprise a first container which includes TNF receptor and a second or more containers comprising anti-TNF antibody and a standard preparation of TNF.
  • Standard TNF receptors may include the monomeric as well as cross-linked dimeric and trimeric forms.
  • Suitable TNF receptors for such kits include 55 kDa human TNF receptor extracellular domain covalently linked to mouse IgG. ⁇ heavy chain or covalently linked to human IgG heavy chain immunoglobulin.
  • Iigand couples in which the receptor consists of a single polypeptide chain.
  • the method could be applied in some instances in which the receptor consists of multiple polypeptide chains.
  • the method would be most applicable in those instances in which a single membrane-spanning domain exists within the receptor sequence (e.g., the receptor does not enter and re-emerge from the cell membrane several times) .
  • the new assay methods are contemplated to have important prognostic value in determining the course of neoplastic or infectious or inflammatory diseases and in influencing therapeutic decisions.
  • Knowledge of levels of certain factors, hormones, cytokines and the like may be harbingers of the course and severity of many diseases, including several types of cancer. Such assays would be frequently and widely applied.
  • TNF is an important mediator of many diseases in man and in animals.
  • the disclosed assay is easily adapted to clinical use in determining TNF levels related to disease state. Other cytokines could be detected in the same manner.
  • the described receptor mediated label transfer assay (RELAY) is readily adaptable to determination of minute amounts of receptor or Iigand in a cell or tissue.
  • Rapid, automated tests will allow sampling of large numbers of samples without the need to obtain large sample volumes or tissue mass.
  • Figure 1 shows a gel shift assay capture of radiolabelled TNF binding protein by increasing concentrations of TNF. Arrows indicate shifted (left) and unshifted (right) TNF binding protein.
  • FIG. 2 shows the construct for TNF receptor binding domain attached to the IgG heavy chain domains C H 2 through C H 3.
  • CTG GTT CCG CGT GGA TCC in Fig.2 is represented by residues 1-18 of SEQ ID NO:4; and Leu Val Pro Arg Gly Ser in Fig.2 is SEQ ID NO:6.
  • FIG 3 shows saturation of TNF by excess of radiolabelled TNF binding protein; +, anti-TNF antibody ( ⁇ !-TNF) conjugated to sepharose beads used for immunoprecipitation; -, control beads (blocked with ethanolamine) used in the system; I + , quantity of radiolabelled TNF binding protein added to the assay system.
  • Figure 4 shows the detection of complexes formed between 125 I-labelled TNF inhibitor and TNF added to 5 ml of human plasma in the quantities indicated. As a marker of the uncomplexed inhibitor, crosslinked, labelled inhibitor was added to the lane marked I* + DSS. Other controls include the omission of antibody ( ⁇ ;TNF) from the sepharose beads, and the omission of exogenous TNF form the system as indicated.
  • ⁇ anti-TNF antibody
  • FIG. 5 shows enhanced sensitivity of complex detection using 131 I-labelled TNF inhibitor.
  • TNF samples were added to 0.5 ml of plasma.
  • a complex is visible with the addition of 0.05 pg of TNF, corresponding to detection at a 200 aM concentration.
  • Controls are the same as in Figure 4.
  • Arrows indicate shifted (left) and unshifted (right) forms of the TNF binding protein.
  • the present invention exploits the extremely high binding specificity of the 55 kDa human tumor necrosis factor (TNF) receptor in an assay designed to detect TNF with sensitivity limited only by limits imposed by the labelling agent detection.
  • Bivalent derivatives of the 55 kDa TNF receptor referred herein as the "TNF binding protein" in which the extracellular domain is coupled to an IgG heavy chain ordinarily bind TNF with very high affinity because of interaction with two separate sites on the trimer surface.
  • the TNF binding protein is radioiodinated to a high specific activity and then added to plasma at a saturating concentration sufficient to bind all active TNF present in the solution.
  • Covalently bound adducts between molecules of TNF and molecules of the binding protein are then produced by crosslinking with disuccinimidyl suberate (DSS) .
  • DSS disuccinimidyl suberate
  • the complexes are swept out of solution using Sepharose beads to which polyclonal anti-TNF antibodies have been affixed.
  • the latter may be removed by washing.
  • As little as 50 fg of active TNF (600,000 trimers) may be detected in a 5 mL sample of plasma using this approach, corresponding to detection of TNF at a 200 aM concentration.
  • no TNF is detectable in normal plasma specimens, indicating that normal plasma contains active TNF at a concentration of less than 200 aM.
  • the disclosed method extends the sensitivity of specific protein assays by several orders of magnitude. It does so without loss in specificity, through a combination of several techniques not previously used for this purpose.
  • the extremely high specificity of the assay derives from the inherent specificity of hormone:receptor interaction. Superior sensitivity is achieved because of conversion of a non-covalent interaction between a hormone and its receptor to a covalent interaction through the use of a cross-linking reagent.
  • the novel method produces a highly specific formation of cross-links of a covalent nature between receptor and Iigand, to the exclusion of cross-links that might form between the receptor and another component of a complex biological solution.
  • Such specific cross linking is possible because of the proximity between macromolecules fostered by the reversible interaction between soluble receptor and its Iigand.
  • the high signal-to-noise ratio afforded by separations using polyacrylamide gel electrophoresis has been exploited, enabling detection of femtogram levels of receptor Iigand complexes.
  • the examples described herein show that the sensitivity of detection of TNF (or conversely, of the TNF receptor) is so great that it is limited only by the sensitivity of detection of the radionuclide that is used as the basis for the assay.
  • This level of sensitivity is 1,000 to 100,000 times greater than that can be achieved through the use of a cytotoxicity assay or ELISA assay.
  • the bivalent TNF biding protein used in these studies was produced by Chinese hamster ovary cells that had been permanently transfected with an expression construct encoding a secreted form of the protein Loetscher, et al.. 1990.
  • the binding protein was purified to homogeneity using both protein G-sepharose and protein A-sepharose.
  • 500 ml of conditioned medium from cells transfected with the inhibitor construct was precipitated with 40% saturated ammonium sulfate, redissolved in 5 ml of water, and dialyzed against 50 mM tris, pH 7.8.
  • the supernatant was loaded onto a protein G-sepharose column (Pharmacia), using a 3:1 ratio of supernatant to bed volume.
  • the column was washed with 0.05 M bicine, pH 8.8. NaCl was then added to the non- retained fraction (containing the TNF inhibitor) , to a final concentration of 3.0 M.
  • the sample was then added to a protein A-sepharose column (1 ml bed volume) .
  • the beads were washed extensively with 0.05 M bicine, pH 8.8, containing 3 M NaCl.
  • the inhibitor was eluted from the column using 0.01 M acetic acid. 200 ⁇ l fractions were collected and immediately neutralized with bicine buffer.
  • Recombinant human TNF was obtained from Genentech, Inc. , and had a specific activity of approximately 2 x 10 7 U/mg protein.
  • Radioiodination was performed according to the method of Fraker and Speck (1978) .
  • a specific activity corresponding to the incorporation of more than one atom of iodine into each dimeric TNF binding protein was routinely achieved without noticeable loss of binding potency.
  • 125 ⁇ was used as the label, since its long half-life made it the more economical and convenient choice. Even greater sensitivity was obtained when 131 ⁇ was used.
  • IgG was isolated from the serum by ammonium sulfate fractionation and Mono Q chromatography. It was then coupled to cyanogen bromide-activated sepharose CL4B, excess sites being blocked by the addition of 1 M monoethanolamine, pH 8.9) . Control beads, lacking antibody, were produced by blocking with monoethanolamine alone (i.e., the beads were not exposed to the antibody preparation) . The IgG content of the beads was estimated at 1.2 mg/mL of packed resin.
  • the TNF assay was performed as follows. Up to 5 mL of heparinized human plasma, to which varying amounts of TNF had been added, was incubated in a polypropylene tube (Falcon plastics, Oxnard, Ca) for 1 hour at 37 C with radiolabelled TNF binding protein, which was added up to the indicated concentration. (If smaller volumes of plasma were used for assay, the crosslinking reagent was decreased in volume proportionately.) The samples were then mixed vigorously using a vortex mixer, and during agitated, 0.25 mL of 0.025 M DSS, dissolved in dimethylsulfoxide, was added. Crosslinking was allowed to occur for 40 minutes, after which the reaction was considered complete.
  • the beads were heated to 100°C, and applied together with the sample buffer to a 8% polyacrylamide gel for electrophoresis. After electrophoresis, the gel was fixed and stained with Coomassie blue. If 131 I was used as the label, the gel was additionally treated with Enlightening (DuPont) prior to drying and autoradiography. Gels were exposed to film for 16 hours at -80°C.
  • the following example illustrates the detection of plasma TNF.
  • the method employs a 131 I-labelled recombinant TNF receptor capture Iigand which is cross- linked to the TNF, then isolated and detected by gel electrophoresis.
  • Heparinized blood was rapidly separated into cellular and plasma components, and the plasma was isolated and maintained in a frozen state until the assay can be performed.
  • a chimeric TNF consisting of the 55 kD human TNF receptor extracellular domain covalently linked to a mouse IgG-1 heavy chain, was used as the capturing Iigand for the detection of TNF. This Iigand was radiolabeled to the maximum specific activity consistent with its retention of biological activity. Approximately 1 iodine atoms may be incorporated per soluble receptor molecule when 131 I is employed.
  • ⁇ g of the labelled soluble receptor (a vast excess with respect to the quantity of TNF present in the plasma sample) was then added to 10 ml of plasma.
  • the sample, together with the labelled soluble receptor was allowed to incubate at room temperature for a period of 60 minutes, during which time virtually all of the biologically active TNF present within the sample was engaged by the labelled soluble receptor.
  • a cross ⁇ linking reagent, disuccinimidyl suberate (DSS) was then added to the plasma to a final concentration of 1 mg/ml .
  • the DSS was permitted to perform covalent cross-links between the soluble receptor and the TNF for a period of 30 minutes.
  • the reaction was terminated by the addition of Tris, pH 8.0, to a concentration of 50 mM.
  • the sensitivity of the assay is illustrated in a system employing 125 I-labelled binding protein. As little as 0.2 pg of TNF may be detected following addition to a 5 mL sample of plasma. This corresponds to the detection of TNF at a concentration of 8 x 10 "16 M. An aliquot of the same plasma sample from which TNF was omitted tests negative in the assay (lane 6) . Omission of anti-TNF antibody from beads added to the system causes a complete loss of signal (lane 7) .
  • Anti-mouse IgG or anti TNF IgG affinity purified and immobilized on agarose beads, was then added to the sample, which was mixed end-over-end for one hour.
  • the beads were sedimented by low-speed centrifugation and washed in 200 ⁇ l of 50 mM Tris, pH 8.0 in order to remove excess human plasma proteins, including immunoglobins and albumin.
  • the beads were then suspended in electrophoresis sample buffer containing SDS, and heated to 100°C in order to liberate the TNF:soluble receptor complexes and unbound soluble receptor (which may exist as a contaminant when anti-TNF is used in the concentration step) , and also to denature the immobilized IgG.
  • the sample was subjected to electrophoresis under denaturing conditions in a polyacrylamide gel .
  • the presence of TNF in the sample was detected by observation of a "shifted" band with slower mobility than the soluble receptor, which migrated under denaturing conditions as a dimer (having been effectively cross-linked) with a molecular weight of approximately 140 kD.
  • the radiolabeled receptor migrated with an apparent molecular weight of approximately 190 kD. If complexed with the TNF dimer, a mobility corresponding to approximately 175 kD was observed.
  • the intensity of the shifted bands indicated the quantity of TNF present in the plasma sample.
  • a phosphorimager may be applied to allow exact measurement of the cross-linked TNF.
  • a Europium screen is exposed to a radioactive source such as a dried gel containing the radiolabelled complex. Bombardment of the Europium results in the emission of light which is read by a detector at the end of the exposure.
  • TNF chimeric construct between the 55 kDa segment of TNF and murine IgG- j ⁇ heavy chain.
  • Cloning of a cDNA Encoding the TNF Receptor Extracellular Domain The TNF receptor extracellular domain (TNFR-ED) was cloned from total cytoplasmic RNA prepared from HL60 cells using a reverse transcriptase/PCR protocol (Kawasaki, 1990) . Briefly, an oligonucleotide primer with the sequence cttaagcttagtactcaTGTGGTGCCTGAGTCCTCAG (SEQ ID NO:l), corresponding to the 3' end of the extracellular domain, was used to direct first-strand cDNA synthesis in a total volume of 20 ⁇ l .
  • the reaction was then diluted to 100 ⁇ l in PCR buffer (Kawasaki, 1990) and a second primer with the sequence gcgcatcgaTCTGGCATGGGCCTCTCCACC (SEQ ID NO:2) , corresponding to the 5' end of the human TNF receptor, was added.
  • the reaction was subjected to 40 cycles of denaturation and synthesis in an automated temperature cycler (Perkin Elmer Cetus Instrs., Norwalk, CT) .
  • the band corresponding to the TNFR-ED was purified by gel electrophoresis and ligated into the vector pGEM- 3Z.
  • a plasmid encoding a murine IgG-1 heavy chain cDNA was obtained from Dr. C. Haseman of the Howard Hughes Medical Institute.
  • the TNFR-ED and the IgG heavy chain cDNAs were separately amplified by PCR using primers gcgcatcgaTCTGGCATGGGCCTCTCCACC (SEQ ID NO:2), corresponding to the 5' end of the TNFR-ED moiety; ggatccacgcggaaccagTGTGGTGCCTGAGTCCTC (SEQ ID NO:3), corresponding to the 3' TNFR-ED moiety and the thrombin cleavage site; ctggttccgcgtggatccGTGCCAGGAGAGTG (SEQ ID NO:4) , corresponding to the thrombin cleavage site and the 5' end of the IgG moiety; and attaagcattctagaTCATTTACCAGGAGAGTG (SEQ ID NO:2)
  • the PCR products obtained after the first synthetic PCR reaction thus carry the thrombin cleavage site on their 3' (TNFR-ED) and 5' (IgG) ends (Fig. 1) .
  • the PCR products were isolated on a low melting point gel and slices containing the correct DNAs were combined and used for a second round of PCR amplification, using only primers corresponding to the 5' end of the TNFR-ED and 3' end of the IgG. This PCR reaction effectively joins the TNFR-ED and the IgG through the thrombin cleavage site.
  • the construct was then digested with Clal and Xbal (encoded in the 5' TNFR-ED and 3' IgG primers) and ligated into the vector pCMV4 (Andersson, et al . , 1989) .
  • the entire sequence was verified by dideoxynucleotide sequencing (Sanger, et al . , 1977) on both strands.
  • TNF TNF-derived neurotrophic factor
  • Human recombinant TNF was iodinated using the iodogen method (Fraker & Speck, 1978) .
  • Radiolabeled TNF was separated from unincorporated 125 ⁇ by chromatography on Sephadex G-25 (Pharmacia Fine Chemicals, Piscataway, NJ) .
  • the specific activity of the final preparation was 10 4 cpm/ng TNF.
  • TNF cross-linking of TNF to Inhibi tor.
  • 25 ng of 125 I TNF was incubated for 1 h at 37°C with 100 ng of purified inhibitor, or inhibitor that had been cleaved with thrombin, in 50 ⁇ l of buffer (25 mM Hepes, pH 7.2, 50 mM NaCl, 0.1% BSA) .
  • buffer 25 mM Hepes, pH 7.2, 50 mM NaCl, 0.1% BSA
  • 2 ⁇ g of unlabeled competitor TNF was included in the reaction; 2.5 ⁇ g of disuccinimidylsuberate (DSS; Pierce Chemical Co., Rockford, IL; 9.3 mg/ml in DMSO) was added and the reaction was incubated for 30 min at 37°C. The reaction was then terminated by the addition of SDS sample buffer.
  • DDSS disuccinimidylsuberate
  • Example 2 The same approach illustrated in Example 1 was used to measure the concentration of soluble TNF receptor present in human plasma. In this case, however, the label was applied to TNF (which was then used as the capture reagent) rather than to the soluble receptor. Using 131 I, as little 10 fg of TNF was detected in 10 ml of human plasma.
  • Example 1 It is contemplated that the method described in Example 1 may be utilized as a standard clinical test for variety of hormones under conditions in which maximum sensitivity is required.
  • TNF may be responsible for certain aspects of the clinical progression of breast cancer and for the hypercalcemia of malignancy as well as wasting, anemia and other clinical problems associated with this disease. Detection of elevated plasma concentrations of TNF may serve as a guide to therapy aimed at neutralizing TNF.
  • chronic inflammatory disease such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, Crohn's disease, ulcerative colitis
  • TNF is thought to function as an important mediator of inflammation.
  • tuberculosis Leishmaniasis, malaria and related parasitic diseases. Detection of TNF may also have prognostic significance and provide a guide to therapy of such diseases.

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Abstract

Nouveau procédé de détection de quantités inférieures au picogramme de récepteurs solubles et de complexes récepteur-ligand. Ledit procédé est extrêmement sensible et a été utilisé pour détecter le facteur de nécrose tumorale dans des échantillons de plasma. Ledit procédé tire profit du pouvoir de résolution de l'électrophorèse en gel de polyacrilamide combinée avec la spécificité conférée par l'interaction récepteur-ligand. On contourne les limitations imposées uniquement par la constante d'affinité de l'interaction entre ligand et récepteur en formant une liaison covalente entre le récepteur et le ligand. Ledit dosage est en principe applicable à la détection de nombreuses hormones protéiques pour lesquelles des récepteurs solubles existent, et convient particulièrement à la détection de protéines qui engagent des sous-unités multiples de récepteur. Ledit procédé représente un perfectionnement important par rapport aux procédés classiques de dosage de protéines à l'état de trace, et permet la détection spécifique de quelques centaines de milliers de molécules actives seulement.
PCT/US1994/009553 1993-08-23 1994-08-23 Dosage par transfert de marqueur induit par recepteur WO1995006257A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043787A1 (fr) * 1999-01-26 2000-07-27 Du Pont Pharmaceuticals Company Tests d'electrophorese permettant d'evaluer les changements de conformation des integrines induits par leur liaison avec un ligand

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. POLTERAK ET AL.: "Receptor-mediated label-transfer assay (RELAY): a novel method for the detection of plasma tumor necrosis factor at atomolar concentrations", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 169, no. 1, 1994, AMSTERDAM NL, pages 93 - 99 *
J.A. CARLINO ET AL.: "Use os a sensitive receptor binding assay to discriminate between full-length and truncated human recombinant tumor necrosis factor proteins", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 262, no. 3, 25 January 1987 (1987-01-25), ROCKVILLE MD USA, pages 958 - 961 *
K. PEPPEL ET AL.: "A tumor necrosis factor (TNF) receptor - IgG heavy chain chimeric protein as a bivalent antagonist of TNF activity.", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 174, 1 December 1991 (1991-12-01), NEW YORK, NY USA, pages 1483 - 1489 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043787A1 (fr) * 1999-01-26 2000-07-27 Du Pont Pharmaceuticals Company Tests d'electrophorese permettant d'evaluer les changements de conformation des integrines induits par leur liaison avec un ligand

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