Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
• • 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
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

• This invention relates generally to monoclonal antibodies which bind to nerve growth factor receptor, and more particularly, relates to an immunoassay and monoclonal antibodies useful for determining the presence of human nerve growth factor receptor or human truncated nerve growth factor receptor in test samples.
• Nerve growth factor (NGF) receptors have been characterized on neuronal cell types and have been categorized into high and low affinity forms of the receptor. It traditionally has been thought that the high affinity form of the receptor mediates the trophic and tropic actions of NGF in neurons, while the significance of the low affinity forms has remained uncertain. The low affinity form of the NGF receptor also has been identified on non-neuronai cells in tissue culture preparations. A. Z. Zimmerman and A. Sutter, Embo J. 2:879-885 (1983); S. T. Carbonetto and R. W. Stach, Dev. Brain Res. 3:463-473 (1982). Defined cultures of highly purified
• NGF-Rt NGF receptor
• NGF receptor truncation also has been demonstrated in vivo, where high levels of NGF-Rt were observed in the urine and plasma of neonatal rats followed by reduced levels in the adult. P. S. DiStefano and E. M. Johnson, Proc. Natl. Acad. Sci USA. Ibid. Furthermore, after sciatic nerve transection in the adult (when Schwann cell NGF receptors are induced) NGF-Rt levels are significantly elevated for prolonged periods in the urine. Coupled with in vitro observations, these developmental and regenerative studies suggest that Schwann cells are the predominant cell type that is shedding NGF-Rt, since NGF-Rt in biological fluids parallels Schwann cell NGF receptor expression.
• an assay to determine the regulation and characterization of NGF-Rt in human urine during the course of development. Such an assay could serve to detect NGF-Rt marker as an indication of abnormal development, regeneration and degeneration of peripheral nerves and qua ⁇ titate levels of NGF-Rt present in a test sample. It also would be advantageous to provide cell lines and monoclonal antibodies raised against human NGF-Rt, which recognize distinct epitopes of the receptor. This assay thus offers the unique advantage that certain monoclonal antibodies described herein recognize distinct epitope(s) on the NGF receptor or truncated NGF receptor molecule, allowing the execution of a two- site assay heretofore not possible with previously existing reagents.
• the present invention provides monoclonal antibodies or fragments thereof which specifically bind to human nerve growth factor receptor and human truncated nerve growth factor receptor, and which also specifically bind to monkey truncated nerve growth factor receptor, and which does not significantly bind to rat or chick nerve growth factor receptor.
• the hybridomas which produce these monoclonal antibodies are identified as follows: IIIG5 (A.T.C.C. deposit No. CRL10617, producing monoclonal antibody IIIG5), VIID1 (A.T.C.C. deposit No.CRL10618, producing monoclonal antibody VIID1), VIHC8 (A.T.C.C. deposit No. CRL10619, producing monoclonal antibody VIIIC8) and XIF1 (A.T.C.C. deposit No. CRL10620, producing monoclonal antibody XIF1).
• the present invention also provides an assay to determine the presence of human nerve growth factor receptor or human truncated nerve growth factor receptor in a test sample, wherein a test sample which may contain human nerve growth factor receptor is contacted with a solid phase to which a monoclonal antibody or fragment thereof which specifically binds to human nerve growth factor receptor and human truncated nerve growth factor receptor, and which also specifically binds to monkey truncated nerve growth factor receptor, and which do not significantly bind to rat or chick nerve growth factor receptor has been bound, to form a mixture. This mixture is incubated for a time and under conditions sufficient for antigen/antibody complexes to form.
• the so-formed complexes then are contacted with an indicator reagent comprising a monoclonal antibody or fragment thereof which specifically binds to human nerve growth factor receptor and human truncated nerve growth factor receptor, and which also binds to monkey truncated nerve growth factor receptor, and which does not significantly bind to rat or chick nerve growth factor receptor has been bound, to form a second mixture.
• This second mixture is incubated for a time and under conditions sufficient for antibody/antigen/antibody complexes to form.
• the presence of human nerve growth factor receptor or human truncated nerve growth factor receptor is determined by detecting the measurable signal generated.
• the amount of human nerve growth factor receptor present in the test sample is proportional to the amount of signal generated.
• the signal generating compound can be selected from the group consisting of luminescent compounds, chemiluminescent compounds, enzymes, and radioactive elements.
• the present invention also provides assay kits for determining the presence and/or amount of human nerve growth factor in test samples.
• FIG. 1 is a photograph of an autoradiograph on SDS-polyacrylamide gel.
• lane 1 Standards are shown in lane 1 and include the following 14 -C-methylated proteins: myosin (200,000), phosphorylase B (92,500), bovine serum albumin (69,000), ovalbumin (46,000) and carbonic anhydrase (30,000).
• myosin 200,000
• phosphorylase B 92,500
• bovine serum albumin 69,000
• ovalbumin 46,000
• carbonic anhydrase 30,000.
• a sample of E9b conditioned medium 100 ⁇ l was incubated with 4 nM 125 I-NGF (lane 2) or radiolabeled NGF in the presence of a 150-fold excess of unlabeled NGF (lane 3).
• FIG. 2 is a photograph of an autoradiograph in which each of the monoclonal antibodies of the invention (5 ⁇ g) were incubated with NGF receptor affinity-cross-
• Standards include the following 14 C-methylated proteins: rriyosin (200,000), phosphorylase B (92,500), bovine serum albumin (69,00), ovalbumin (46,000) and carbonic anhydrase (30,000).
• FIG. 3 is a photograph of an autoradiograph in which each of the monoclonal antibodies of the invention was incubated with 125 l affinity labeled NGF-Rt (2.2 ⁇ g protein) purified approximately 650-fold from E9b conditioned medium by chromatography on ME20.4 sepharose and affinity labeled with 125 I-NGF. Standards are those described in FIG. 2.
• FIG. 4 is a photograph of an autoradiograph in which each of the monoclonal antibodies of the invention were incubated with 125 I-NGF cross-linked NGF-R contained in solubilized preparations from SH-SY5Y cells.
• the standards are those described in FIG. 2.
• FIGS. 5A-B are photographs of immunoblots of E9b cell NGF-R under non- reducing and reducing conditions.
• FIG. 5A is a photograph of the solubilized preparation loaded directly on a non-reducing 10% SDS-polyacrylamide gel followed by electrophoresis and transfer to nitrocellulose membrane (non-reduced).
• FIG. 5B is a photograph of the immunoblot of samples mixed with 5% beta- mercaptoethanol and boiled prior to resolution on gels and immunoblotting (reduced).
• FIGS. 6A-6D are bar graphs of competition studies wherein the counts (cpm x 10- 3 /weII) of each monoclonal antibody labeled with 125 l were plotted against NGF-Rt bound to immobilized unlabeled antibody, as indicated on the graphs.
• FIG. 6A is a graph of 1 25 I-IIIG5.
• FIG. 6B is a graph of 12 5
• FIG. 6C is a graph of 1 5
• FIG. 6D is a graph of 125
• FIGS. 7A-B are bar graphs of two-site RISA using 125 l-labeled IIIG5 monoclonal antibody of the invention to detect NGF receptor and NGF-Rt bound to immobilized antibodies. Data are expressed as mean +/- SD. An average for the background binding when either Ltk- solubilized cells or conditioned medium was used in the assay is shown by the dotted line ( ).
• FIG. 7A shows E9b cells (hatched bar) that were solubilized and use as a source of NGF receptor; Ltk- solubilized preparation (open bar) were examined in parallel to assess non-specific binding.
• FIG. 7B shows E9b conditioned medium (solid bar) used as an assay source of NGF-Rt. Conditioned medium (open bar) was examined in parallel to assess non ⁇ specific binding.
• FIG. 8A is a graph of the linearity of binding of 125 I-IIIG5 to increasing amounts of NGF-Rt immobilized on a solid support by antibody XIF1.
• FIG. 8B is a graph of the correlation of relative values obtained for NGF-Rt in serial dilutions of E9b conditioned medium concentrated by ammonium sulfate precipitation using the two-site RISA assay of the invention and the CLIP assay. Data in each assay are expressed as a percent of the maximum value obtained for NGF-Rt.
• FIG. 9A is a graph of the counts per minute of 1 2 5
• FIG. 9B is a graph of the counts per minute of 125 I-IIIG5 bound per well versus urine volume added to the assay.
• Urine sample from a 6-year old male was diluted with HEPES to a final volume of 50 ⁇ l.
• FIG. 10 is a graph of urine NGF-Rt plotted as a function of age in humans.
• FIG. 11 is a graph of urine NGF-Rt versus age, expressed as per mg urine protein.
• the present invention provides novel cell lines (hybridomas) which produce (secrete) monoclonal antibodies to NGF receptor, immunoassays which use the monoclonal antibodies, and kits which contain these monoclonal antibodies.
• These cell lines are identified as cell line IIIG5 which produces monoclonal antibody IIIG5, cell line VIID1 which produces monoclonal antibody VIID1 , cell line VIIIC8 which produces monoclonal antibody VIIIC8, and cell line XIF1 , which produces monoclonal antibody XI F1.
• cell line IIIG5 which produces monoclonal antibody IIIG5
• cell line VIID1 which produces monoclonal antibody VIID1
• cell line VIIIC8 which produces monoclonal antibody VIIIC8
• cell line XIF1 which produces monoclonal antibody XI F1.
• the monoclonal antibodies of the invention can be employed in various assay systems to determine the presence, if any, of truncated NGF receptor in a test sample. Fragments of the monoclonal antibodies also can be used.
• the present invention provides an assay to detect human NGF receptor and/or truncated NGF- receptor. We have discovered a distinct developmental regulation of NGF receptor in human urine which is similar to that seen in the rat. P. S. DiStefano and E. M. Johnson, Proc. Natl. Acad. Sci USA. Ibid. No sexual dimorphism was evident at any time during development, nor was there any diurnal variation associated with NGF receptor truncation in the adult.
• NGF receptor truncation correlates well with the development of function in peripheral nerves, strengthening the hypothesis that the predominant cell type shedding NGF receptor is the Schwann cell. Furthermore, NGF receptor in test samples such as biological fluids can serve as a biochemical marker for abnormal development, regeneration and degeneration of peripheral neurons.
• a capture reagent comprising a first monoclonal antibody or fragment thereof which specifically binds to a distinct epitope of NGF receptor and truncated nerve growth factor receptor, which has been coated on a solid phase, is contacted with a test sample which may contain either or both NGF receptor and truncated NGF receptor, to form a mixture.
• a test sample which may contain either or both NGF receptor and truncated NGF receptor
• This mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes.
• an indicator reagent which comprises a second monoclonal antibody or fragment thereof specific to a NGF receptor and truncated nerve growth factor receptor to which a signal generating compound has been attached, to form a second mixture.
• This second mixture is incubated for a time and under conditions sufficient to form antibody/antigen/a ⁇ tibody complexes.
• the presence of NGF receptor and/or truncated nerve growth factor receptor in the test sample which is captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating compound.
• the amount of NGF receptor present is proportional to the signal generated.
• the monoclonal antibody designated XIF1 be used as the capture antibody, and that monoclonal antibody designated as IIIG5, which recognizes a distinct and different epitope of the NGF receptor or truncated NGF receptor molecules compared to XIF1 , be used as the indicator reagent.
• a monoclonal antibody or combination of monoclonal antibodies, or fragment(s) thereof, which specifically bind to NGF receptor or truncated NGF receptor which are bound to a solid support, the test sample, and an indicator reagent comprising a monoclonal antibody or combination of monoclonal antibodies, or fragment(s) thereof, which specifically bind to NGF receptor to which a signal generating compound is attached, are contacted to form a mixture.
• This mixture is incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes.
• the presence, if any, of NGF receptor in the test sample and captured on the solid phase is determined by detecting the measurable signal generated by the signal generating compound.
• the amount of NGF receptor present in the test sample is proportional to the signal generated.
• one or a combination of more than one monoclonal antibody of the invention or fragment thereof is employed as a competitive probe for the detection of antibodies to NGF receptor and/or truncated NGF receptor.
• NGF receptor and/or truncated NGF receptor can be coated on a solid phase.
• a test sample suspected of containing antibody to NGF receptor and/or truncated nerve growth factor receptor then is incubated with an indicator reagent comprising a signal generating compound attached to one or a combination of more than one monoclonal antibody of the invention or fragment thereof, for a time and under conditions sufficient to form antigen/antibody/complexes of either the test sample and indicator reagent to the solid phase or the indicator reagent to the solid phase.
• the reduction in binding of the monoclonal antibody to the solid phase can be quantitatively measured.
• a measurable reduction in signal compared to the signal generated from a confirmed negative NGF receptor and truncated NGF receptor test sample indicates the presence of anti-NGF receptor antibodies and/or anti-truncated NGF receptor antibodies in the test sample.
• each of the monoclonal antibodies of the present invention can be employed in the detection of NGF receptor and/or truncated NGF receptor in fixed or fresh tissues or cells by immunochemical analysis.
• these monoclonal antibodies can be bound to matrices and used for affinity purification of specific NGF receptor and truncated NGF receptor proteins from cell cultures.
• These monoclonal antibodies or fragments thereof of the present invention can be provided individually to detect NGF receptor and/or truncated NGF receptor.
• Combinations of the monoclonal antibodies (and fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of anti-NGF receptor antibodies, with different binding specificities.
• polyclonal antibodies to NGF receptor and truncated NGF receptor may be used as either the capture reagent or indicator reagent in all the assays described herein.
• the polyclonal antibody or fragment thereof which may be used in the assay formats should specifically bind to NGF receptor and/or truncated nerve growth factor receptor.
• the polyclonal antibody used can be derived from avian, mammalian or reptilian origin; thus, human, goat, rabbit or sheep anti-NGF receptor and/or anti-truncated NGF receptor polyclonal antibody can be used.
• the polyclonal antibodies which may be employed in the assays can be used either alone or as a cocktail of polyclonal antibodies.
• Test samples which can be tested by the methods of the present invention described herein include biological fluids such as urine, whole blood, plasma, serum, cerebrospinal fluid, saliva, sweat, semen, or conditioned medium of cultured human cells. It also is contemplated that cells and tissues which are fixed or fresh can be employed.
• Solid supports are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, Sepharose-like beads, and others.
• the indicator reagent comprises a signal generating compound (label) which is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for NGF receptor and/or truncated NGF receptor.
• label a signal generating compound
• Specific binding member means a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule.
• the indicator reagent in addition to being an antibody member of a specific binding pair for NGF receptor and/or truncated nerve growth factor receptor, the indicator reagent also can be a member of any specific binding pair, including either hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
• hapten-anti-hapten systems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and the like.
• the immunoreactive specific binding member can be an antibody, an antigen, or an antibody/antigen complex that is capable of binding either to NGF receptor and/or truncated NGF receptor as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay.
• the various signal generating compounds (labels) contemplated include chromogens, catalysts such as enzymes, luminescent compounds such as fluorescein and rhodamine, chemiluminescent compounds, radioactive elements, and direct visual labels.
• enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like.
• radioactive elements include 1 25 l, 3 H and 35 S. The selection of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
• the reagent(s) employed for the assay can be provided in the form of a kit with one or more containers such as vials or bottles containing a separate reagent such as a monoclonal antibody
• Example 1 Immunization/Cell Fusion A partially purified preparation of NGF-Rt was prepared for use as an immunogen as follows. E9b cells were grown as described by M. V. Chao et al.. Science 232:518-521 [1986]). Conditioned medium was decanted from the cells, brought to 0.02% with sodium azide and stored at 4°C. An immunoaffinity chromatography resin was prepared by coupling affinity purified monoclonal antibody ME20.4 (A. H. Ross et al.. Proc. Natl. Acad. Sci. USA 81 :6681-6685
• IV injection of approximately 40 ⁇ g total protein was administered seven (7) weeks after the three (3) week boost.
• the hybridomas were maintained in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum (FCS), glutamine (2 mM), sodium pyruvate (1 mM), nonessential amino acids (10 mM), 2-mercaptoethanol (50 ⁇ M) and n-(2- hydroxyethyI)1piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3 (10 mM).
• FCS fetal calf serum
• glutamine (2 mM
• sodium pyruvate (1 mM
• nonessential amino acids 10 mM
• 2-mercaptoethanol 50 ⁇ M
• HEPES n-(2- hydroxyethyI)1piperazine-N'-2-ethanesulfonic acid
• Immulon 2 Removawell strips (available from Dynatech Labs, Alexandria, VA) were coated with goat anti-mouse IgG (50 ⁇ l, 50 ⁇ g/ml) in PBS (pH 8.0) overnight at 4°C or at room temperature for one (1 ) hour. After removal of the goat anti-mouse IgG, the wells were blocked with 1.5% BSA (150 ⁇ g/ml) in PBS for 30 minutes at room temperature. The wells were washed three (3) times with cold PBS followed by the addition of hybridoma supematants (50 ⁇ l) and incubation for approximately two hours at room temperature.
• the wells were washed three (3) times with cold PBS, placed on ice, and 50 ⁇ l of NGF-R cross-linked to 1 25 I-NGF (approximately 25,000 cpm) was added to each well and incubated for 45 minutes at 4°C.
• the wells were washed four (4) times with cold PBS containing 0.05% Tween-
• Receptor-containing samples affinity labeled with 1 25 I-NGF (50 to 100 ⁇ l) were incubated with hybridoma supernatant (50 to 100 ⁇ l), mouse serum (50 ⁇ l of a 1 :100 dilution in PBS) or purified monoclonal antibodies (5 ⁇ g) for two to four hours at 4°C.
• a suspension of goat anti-mouse IgG Sepharose in PBS was added (10%, v/v), and the mixture was incubated for one hour.
• Sepharose in tubes containing NGF-R was washed twice with 500 ⁇ l of PBS containing 0.05% Tween- 20, whereas Sepharose in tubes containing NGF-Rt was washed with PBS containing 0.5% bovine serum albumin (BSA), 0.5% sucrose and 0.1% Tween-20®.
• BSA bovine serum albumin
• Membranes were incubated for one hour at room temperature in PBS containing 5% non-fat dry milk, followed by one rinse with PBS containing 0.5% non-fat dry milk, and incubation for two hours with affinity purified monoclonal antibody (25 ⁇ g/ml) in the same buffer. Membranes were rinsed three times each for five minutes with PBS containing 0.5% non-fat dry milk and 0.05% Tween-20. Membranes then were incubated for two hours in the presence of 1 2 5
• Affinity purified antibodies were radiolabeled with 25 l-Bolton-Hunter reagent.
• Cells (E9b or Ltk-) were solubilized as described hereinabove.
• NGR-Rt in E9b cell conditioned medium was used directly or was purified by immunoaffinity chromatography on a resin constructed using an antibody (ME20.4) to the human NGR-R.
• the column was washed sequentially with PBS, 20 mM sodium phosphate buffer (pH 7.4) containing 0.55 M NaCI, PBS, 50 mM CAPS buffer (pH 9.8).
• NGF- Rt was eluted from the column using CPAS buffer (pH 11.5). Column fractions were brought to pH 7.4 by the addition of 1 M HEPES buffer, pH 7.0.
• Immulon 2 Removawells were coated with 50 ⁇ l of a given monoclonal antibody of the invention (50 ⁇ g/ml) in PBS overnight at 4°C. Additional binding sites were blocked with the addition of 150 ⁇ l of 1.5% BSA in PBS for 30 minutes at room temperature. The wells were washed three times with cold PBS, after which 50 ⁇ l of the receptor-containing preparation was added and incubated for 90 minutes at room temperature. The wells were washed three times with cold PBS followed by the addition of 50 ⁇ l of a given 1 2 5
• Example 8 General Procedures Protein concentration was determined by the method of M. M. Bradford, Anal. Biochem. 72:248-3454 (1976) using crystalline BSA as a standard. Laser densitometry was performed using an LKB UltroScan XL laser densitometer.
• Antibodies were affinity purified from hybridoma supernatant using a protein A- Sepharose monoclonal antibody purification system (Biorad Labs, Richmond, CA). Antibodies were isotyped using a ScreenTypeTM kit obtained from Boehringer Mannhein (Indianapolis, IN).
• NGF-Rt from E9b conditioned medium was affinity labeled with 12 5
• NGF- Rt was immunopur ⁇ fied from E9b conditioned medium and used to immunize a BALB/cByJ mouse. Serum from a mouse receiving a primary immunization and one booster injection of partially purified NGF-Rt was positive for antibody activity to intact NGF-Rt by RISA and immunoprecipitation assay. Spleen cells from this mouse were fused with NS-1 mouse myeloma cells.
• Hybridomas which secreted antibody to the NGF-R were first identified using the RISA assay as described in Example 3. Wells with a signal two times above background radioactivity (34/1056 wells) were rescreened by immunoprecipitation, and 18 of 34 wells remained positive for the presence of antibody to the NGF-R using this assay. Five cell lines continued to screen positive for antibody to NGF-R after expansion of the cell lines, and these were cloned by limiting dilution. After cloning and expansion, four hybridoma lines remained positive for antibody to NGF-R.
• hybridomas are identified as cell line IIIG5 which produces monoclonal antibody IIIG5, cell line VIID1 which produces monoclonal antibody VIID1 , cell line VIIIC8 which produces monoclonal antibody VIIIC8, and cell line XIF1 , which produces monoclonal antibody XIF1.
• the four monoclonal antibodies produced were capable of immunoprecipitating a protein with an apparent molecular weight of 63,000 daltons from a preparation containing affinity labeled NGF-Rt, as shown in FIG. 3. Again, subtracting a monomer of NGF yields a net molecular weight of approximately 50,000 daltons for the truncated form of the receptor. The 125 l-NGF-NGF-Rt complex was not immunoprecipitated by the antibody MOPC21.
• the CLIP assay as described in Example 4 was performed using affinity labeled NGF-R solubilized from SH-SY5Y cells.
• SH-SY5Y cells are a clonal affinity form of the NGF-receptor (see K. H. Sonnenfeld and D. N. Ishii. J * Neurosci. 5:1717-1728 [1985]).
• All four monoclonal antibodies of the invention, as well as the antibody ME20.4 immunoprecipitated the 90,000 Mr NGF-R 1 25 l- NGF-R complex from SH-SY5Y cells receptor, as shown in FIG. 4. No labeled material was immunoprecipitated by MOPC21.
• Example 11 Species Cross- Reactivity Using the two-site RISA as described in Example 3, all of the monoclonal antibodies of the invention bound to NGF-Rt from monkey urine. However, antibodies did not bind to NGF-R from chick embryonic dorsal root ganglia, rat superior cervical ganglia or PC12 cells.
• Example 13 Epitope Mapping and Selection of Monoclonal Antibodies for Assay Solid phase competition studies were performed to determine whether the monoclonal antibodies of the invention bound to distinct receptor epitopes. All of the monoclonal antibodies were tested on the solid phase, as well as serving as the radiolabeled (top) monoclonal antibody. In this assay, the retention of radiolabel in the well indicated that the top and bottom antibody recognized distinct epitopes on the receptor protein. The only exception to this occurred when a single antibody bound to repeated epitopes of the receptor.
• Example 14 Sample Collection and Preparation Urine samples were collected from 70 normal human subjects ranging in age from 1 month to 68 years. Urine from 4 pregnant women in their third trimester
• Urine was collected in polypropylene specimen containers (available from Scientific Products, McGaw Park, IL), immediately placed on ice, and frozen at -80°C within 2 hours of collection. Urine samples were routinely assayed within 2 weeks of collection. There was no reduction in assay values for NGF-Rt or for creatinine when samples were frozen at -80°C for at least three weeks when compared to fresh urine. For neonates, urine was collected from cloth diapers or by the use of U-bags (available from Hollister, Kirksville, MO).
• samples were thawed at 4°C, centrifuged at 13,000 x g for 5 minutes, and diluted 1 :1 with 0.5M HEPES buffer, pH 7.0, before addition to the assay plates as described hereinbelow.
• Assay blanks used as negative controls consisted of 20 mM phosphate, 160 mM NaCI (phosphate buffered saline [PBS]) diluted 1 :1 with HEPES buffer.
• RISA radiometric immunosorbent assay
• NGF-R nerve growth factor receptor
• NGF-Rt truncated nerve growth factor receptor
• XIF1 was chosen as the anchoring (capture) antibody and was used to bind NGF-R from solubilized cell preparations or NGF-Rt to the solid phase.
• -MG5 (5 x 10 6 cpm/well) was used in the final incubation step in the indicator reagent to detect the presence of NGF receptor.
• the assay of the invention is a modification of a radiometric immunosorbent assay described by Pierce et al.. Anal. Biochem. 153:67-74 (1986).
• Monoclonal antibody XIF1 was diluted to 50 ⁇ g/ml in PBS (pH 8.0), and then added to Immulon II Removawell strips (available from Dynatech, Chantilly, VA) in a volume of 50 ⁇ l, and incubated overnight at 4°C. Non-reacted sites on the wells were blocked with 1.5% bovine serum albumin for one (1) hour, and washed with PBS (pH 8.0) using a Dynatech plate washer (Dynawasher IITM, available from Dynatech, Chantilly, VA).
• II1G5 was iodinated to specific activities of 600-800 cpm/fmol and binding was performed at various concentrations of labeled IHG5 for 45 minutes as described herei ⁇ above.
• KD and Bmax values were determined by Scratchard plot. Bmax was expressed as nanograms (ng) 125 I-NGF bound per ⁇ g of creatinine which had been determined as described in Example 15.
• NGF-Rt values obtained by following the procedures described in Examples 16 and 17 from various groups, were determined using one-way ANOVA followed by Newman-Keuls' post-hoc analysis. Significant differences were determined at the .01 level. Binding constants for 1 25 I-NGF binding to NGF-Rt were compared using the Student's t-test.
• Example 16 It was found that the two-site RISA described in Example 16 represented a rapid and reliable means to quantify NGF-Rt levels in human biological fluids.
• the assay was linear with respect to the amount of purified recombinant truncated receptor up to 50,000 cpm.
• the internal standard of E9b conditioned medium routinely registered 10,000 cpm.
• the assay also was linear with the amount of urine added (diluted 1 :1 with HEPES buffer) up to a volume of 25 ⁇ l.
• the sample-to-blank ratios ranged from 3:1 to 30:1, depending on the content of NGF-Rt in the sample.
• Analytical precision was 2- 8% and inter-assay variability was less than 4%, as determined on the urine from
• NGF-Rt in adult urine was found to vary by only 5% over the course of six months.
• the NGF-Rt protein was found to be very stable in urine such that samples could be stored for at least two months at -80°C without experiencing loss of activity. It further was determined that urine samples could be left at room temperature for up to eight hours without loss of NGF-Rt activity or creatinine level. Boiling of urine samples, however, abrogated activity without affecting creatinine levels.
• SUBSTITUTE SHEET Example 16 is such that 65 samples can be assayed in quadruplicate by an individual in one day.
• FIG. 10 shows an expanded x-axis plot of the data in order to gain an estimation of the decrease in urine NGF-Rt during very early development.
• urine NGF-Rt declined rapidly in urine.
• the decline was noticeably more gradual, suggesting a bimodal decrease in urine NGF-Rt during the first four years of life.
• Urine NGF-Rt also was expressed as ng/mg urine protein, as NGF-Rt represents a sizable protein excreted in urine.
• FIG. 11 shows a similar developmental regulation of NGF-Rt when expressed per mg protein, except that the differences between neonate and adult were not as robust. In addition, the variability in the data was much greater when expressed per mg protein compared to creatinine normalization. Regardless of how the data were normalized, however, a similar developmental pattern was evident for NGF-Rt in human urine.
• Urines were sampled from women at 7.5-8.5 months of pregnancy. ** p ⁇ .01 compared to all other groups.
• NGF-Rt Monoclonal antibodies have been produced against a soluble, truncated form of the human NGF-R elaborated by E9b cells. NGF-Rt from E9b cells is similar to
• NGF-Rt found in the conditioned medium of cultured Schwann cells (P. S. DiStefano and E. M. Johnson, Proc. Natl. Acad. Sci. USA 85:270-274 [1988]) and melanoma cells (A. A. Zupan et al., J. Biol. Chem. 264:11714-11720 [1989]) with respect to molecular weight and displacement of 5 I-NGF binding by unlabeled NGF.
• the four monoclonal antibodies of the invention immunoprecipitate affinity labeled receptor species to varying degrees, it is evident that they all recognize the whole (cell surface) and truncated forms of the NGF-R.
• the monoclonal antibodies of the invention produced in this study were screened on the basis of their ability to bind human NGF-R affinity labeled with 12 5
• the monoclonal antibodies of the invention all cross-react with truncated NGF receptor from monkey but not rat or chick.
• the monoclonal antibodies of the invention specifically bind to human nerve growth factor receptor and human truncated nerve growth factor receptor, and also specifically bind to monkey truncated nerve growth factor receptor, and do not significantly bind to rat or chick nerve growth factor receptor.
• the four novel monoclonal antibodies of the invention which bind to distinct epitopes on the NGF-R have allowed the development of the highly sensitive assay of the invention which can be used to measure NGF receptor or truncated nNGF receptor in large numbers of samples.
• the assay of the invention also can be used to measure whole NGF-R extracted from a variety of sources. Measurement of NGF receptor using the two-site RISA offers an added advantage over previously described methods, because NGF receptor can be assayed in the absence of ligand (NGF). Measurement of

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