JPH06503722A - Immunoassays and monoclonal antibodies useful for detecting terminally deleted nerve growth factor receptors - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Immunoassays and monoclonals useful for detecting terminally deleted nerve growth factor receptors antibody Background of the invention This invention generally relates to monoclonal antibodies that bind to nerve growth factor receptors; More specifically, human nerve growth factor receptor or terminal deletion (trun) in the test sample. Immunoassay Useful for Determining the Presence of Human Nerve Growth Factor Receptor B. and monoclonal antibodies.

Nerve growth factor (NGF) receptors have been characterized in neuronal cell types. They are classified into high-affinity receptors and low-affinity receptors. The high affinity receptor is Trophic and tropic effects of NGF on neurons ndtropic actions), while the significance of low-affinity receptors It has traditionally been thought that there is no certainty that The low affinity NGF receptor also , has also been identified in non-neuronal cells in tissue culture preparations. Chinmarma (A, Z.

Z immerman) and Santa (A, 5utter), Embo J , 2 + 879-885 (1983); Carbonet (S, T, Carb onetto) and Stank (R, W, S touch), Dev, Brai n Res, 3: 463-473 (1982), on the surface of Tuyuan cells. Determination of highly purified shikotsuyuan cells to demonstrate the presence of low affinity NGF binding sites Defined cultures are used. Sistef George (P, SD] S tefano) and Johnson (E, M, Joh Nson), J. Neurosci, 8: 231-241 (198 8); Alasuline (J, G, As5ouline) and Pantalice ( J, N, Pantazis), Exp, Ce1l Res, 182: 499-51-512 (1989). In vivo, NGF binding sites are located in axons. Appears in rat Schwann cells in response to cleavage. Tan1uch (M, Tan1uch i) et al., Proc, Natl, Acad, Sci, U.S.A. 83 : 4094-4098 (1986). In addition, the axoneme-Tsuyuan cell contact has not yet been established. During early development, high levels of NGF receptors are localized in peripheral nerves; Disruption of the axon-Tzuwann cell contact results in high levels of NGF reception on the Schwann cell surface. This suggests that it is a physical expression. Developing or regenerating axons are exposed to NGF receptor receptors. proteins (e.g., Taniuchi et al., supra, and dystephages and ch. Rune (D, M, Chelsea) Soc, Neurosci, Abst r, 14:1268 [1988]) and its mRNA (astragalus (astragalus) G, Lemke) and M, Chao, Development 102:499-504 [1988]). NGF receptors are regulated in Schwann cells by physical contact with axonal elements The assumption is that

The present inventors recently demonstrated that Tsuyuan cells maintained in tissue culture have a terminally deleted form. The NGF receptor (NGF-Rt) was shown to be secreted into the medium. Dystepha George and Nelson, Proc, Natl, Acad, Sci, USA 85: 270-274 (1988). Although the number of Tsuyuan cells is not large, , rat chromaffin cell tumor (PCl3) (Nstephage and Johnson , Proc, Natl, Acad, Sci.

USA, top I! Schwannoma (distephage and schwannoma) in rats Zup (A, A, Zup) and human myeloma (A875) (A, A, Zup) an) et al., JBiol, Chem, 264:11714-11720 (19 Some NGF receptor-bearing cells, including 89)), secrete NGF-Rt into the medium. Ru. Interestingly, NGF-dependent neurons of the supraneuroid ganglia have high molecular weight or Secretes only the receptor as a whole. (Dystepher) and Johnson, Pr. oc, Natl, Acad, Sci, USA, above). NGF receptor truncation has also been observed in vivo, with high levels of Bell's NGF-Rt was observed in the urine and plasma of newborn rats, and then Levels in the body decrease. Distephage and Johnson, Proc, Na tl, Acad, Sci, IJ S A, above. Furthermore, in adults ( When the sciatic nerve is transversely cut (when the Ewann cell NGF receptor is induced), N GF-Rt levels are significantly elevated in urine over long periods of time. In vitro observation Taken together with the current research, these developmental and regenerative studies suggest that Tuyuan cells are highly susceptible to NGF-R. This suggests that this is the main cell type that secretes T. Because biological flow This is because NGF-Rt in the body parallels the expression of NGF receptors in Tuyuan cells. Ru.

To determine the regulation and characterization of NGF-Rt in human urine during development. It would be advantageous to provide an assay for. Such assays can detect peripheral nerve Detecting and testing NGF-Rt-mamma as an indicator of normal development, regeneration and degeneration It can be useful in quantifying the level of NGF-Rt present in a sample.

In addition, it recognized a specific epitope of the receptor and was produced against human NGF-Rt. It would also be advantageous to provide monoclonal antibodies and cell lines. Therefore, this The assay allows certain monoclonal antibodies described herein to be used to target the NGF receptor or or by recognizing a specific epitope on the terminally deleted NGF receptor molecule. 2-site assay that could not be performed using existing reagents It offers the unique advantage of being able to perform

Summary of the invention The present invention is particularly directed to human nerve growth factor receptors and terminally deleted human nerve growth factor receptors. It specifically binds to the terminally deleted monkey nerve growth factor receptor, and or a monoclonal antibody that does not significantly bind to the chicken nerve growth factor receptor. provides a snippet of it. Noibridomas that produce these monoclonal antibodies is identified as follows: I [IG 5 (A, T, C, C, accession number No. CRL10617, monoclonal antibody 1 [producing IG5], ■DI (A, T, C, C accession number CRLIO618, produced monoclonal antibody ■D1), ■ C8 (A, T, C, C accession number CRL10619, monoclonal antibody ■C8 production) and XIFI (A, T, C.

C accession number CRL10620, producing monoclonal antibody XIFI).

The present invention relates to human nerve growth factor receptor or terminally deleted human nerve growth factor in a test sample. provides an assay for determining the presence of a child receptor; specific for human nerve growth factor receptor and terminally deleted human nerve growth factor receptor. It also specifically binds to the terminally deleted monkey nerve growth factor receptor. is a monoclonal antibody that does not significantly bind to chicken nerve growth factor receptor or The solid phase to which the fragments are bound contains a sample that may contain the human nerve growth factor receptor. The test sample is contacted to form a mixture. This mixture forms an antigen/antibody complex. Incubate for a time and under conditions sufficient to Then, thus forming The human nerve growth factor receptor and the terminally deleted human nerve growth factor receptor It specifically binds to the human body and also specifically binds to the terminally deleted monkey nerve growth factor receptor. A monoclonal antibody that does not bind significantly to chicken or chicken nerve growth factor receptors. contact with an indicator reagent comprising the body or a fragment thereof to form a second mixture. this the second mixture for a time and under conditions sufficient to form antibody/antigen/antibody complexes. Incubate. Human nerve growth factor receptor and terminally deleted human nerve growth factor The presence of the receptor is determined by detecting the measurable signal produced.

The amount of human nerve growth factor receptor present in the test sample and therefore captured on the solid phase The amount of human nerve growth factor receptor is directly proportional to the amount of signal generated. signal The produced compounds include luminescent compounds, chemiluminescent compounds, enzymes, and radioactive components. It can be selected from a group consisting of elements.

The present invention also provides methods for determining the presence and/or amount of human nerve growth factor in a test sample. We also provide assay kits for testing.

Brief description of the drawing Figure 1 is a photograph of an autoradiograph on a 5DS-polyacrylamide gel. . Standards are shown in lane 1 and include the following I40-methylated proteins: myo Shin (200,000), phosphorylase B (92,500), orchid serum Albumin (69,000), egg albumin (46,000) and carbonyl Quanhydrase (30,000). For E9b conditioning, a sample of the medium (100 μ+ ) with 4 μM + 25 I-NGF (lane 2) or with a 150-fold excess of Incubated with radiolabeled NGF in the presence of labeled NGF (lane 3). Ta.

Figure 2 shows that each monoclonal antibody of the present invention (5 μg) was mixed with l”1-NGF (10 μg). NG affinity cross-linked to 0 μm) and solubilized from E9b cells (3X10') Photograph of an autoradiograph incubated with F receptor. to standard contains the following +4 (-methylated protein: myosin (200,000) , phosphorylase B (92,500), bovine serum albumin (69,000) ), egg albumin (46,000) and carbonic anhydrase (30,000) 000).

Figure 3 shows how each monoclonal antibody of the present invention was cloned on ME20.4 Sepharose. E9b conditional was purified approximately 650 times from the medium by chromatography, and Hfi NG +251 affinity labeled NGF-Rt (2, This is a photograph of an autoradiograph incubated with 2 μg protein). Ru.

The standard is the same as described in FIG.

Figure 4 shows the solubilization preparation of each monoclonal antibody of the present invention from 5H-8Y5Y cells. Incubated with +251-NGF cross-linked NGF-R contained in the product. This is a photo of an autoradiograph. The standard is the same as described in FIG.

Figures 5A-B show immunohistochemistry of NGF-R in E9b cells under non-reducing and reducing conditions. This is a photo of Noplot.

Figure 5A shows the results of loading directly onto a non-reducing 10% 5DS-polyacrylamide gel and then is a photograph of a solubilized preparation subjected to electrophoresis and transfer to a nitrocellulose membrane. (non-reducible).

Figure 5B. Dissociation and immobilization on a gel mixed with 5% β-mercaptoethanol. Photograph of an immunoplot of a sample that was boiled before blotting (reduced ).

Figures 6A-6D show each monoclonal antibody labeled with 1'I as shown on the graph. body counts (cpmxlo-3/well) of NG coupled to immobilized unlabeled antibodies. Figure 2 is a bar graph of a competitive study plotted against F-R.

FIG. 6A is a graph of +251-I[IO2.

FIG. 6B is a graph of +251-■D1.

FIG. 60 is a graph of +'I-■C8.

FIG. 6D is a graph of +251-XIFI.

Figures 7A-B show that the 125I-labeled I[[G5 monoclonal antibody of the present invention was used to Two sites for detecting NGF receptor and NGF-Rt bound to immobilized antibodies It is a bar graph of R15A. Data are expressed as mean +/-3D. Ah background when using Ltk-solubilized cells or conditional medium. The average of the round combinations is shown by the dotted line (,,).

Figure 7A shows solubilized E9b cells (hatched bars) and NGF receptor source. The Ltk-solubilized preparation (birch) was also used to assess non-specific binding. I looked it up at the same time.

Figure 7B shows the E9b conditioned medium (plain) used as the assay source for NGF-Rt. bar) is shown. Conditional medium (birch) was also prepared at the same time to assess non-specific binding. Beta.

Figure 8A shows increasing amounts of NGF-Rt immobilized on a solid support by antibody XIFI. FIG. 2 is a graph showing the linearity of binding of 1251-I[[G5 to.

Figure 8B shows that the sulfur E9b conditioned concentrated by acid ammonium precipitation showed NG in serial dilutions of the medium. It is a graph which shows the correlation of the relative value obtained with respect to F-Rt. for each assay The data in are expressed as a percentage of the maximum value obtained for NGF-Rt. It is.

Figure 9A shows n of terminally deleted purified recombinant human receptor protein (rhNGF-Rt). Count of 12'T-I [[G5 bound per well for g This is a graph that was cut.

Figure 9B shows +257 bound per well versus the amount of urine added to the assay. 1 [This is a graph plotting G5 count 7 minutes. HE performed a urine sample from a 6-year-old boy. Diluted with PES to a final volume of 50 μm.

Figure 10 is a graph plotting urinary NGF-Rt as a function of age in humans. It is. The insert shows a horizontal wide x-axis semi-logarithmic plot of urine NGF-Rt against age. vinegar. The value at 1=0 was determined by extrapolation to the y-axis. The value is 1=0 Expressed as a number.

Figure 11 is a graph of urinary NGF-Rt versus age, per mg urinary protein. It is expressed as follows.

Detailed description of the invention The present invention provides novel cells that produce (secrete) monoclonal antibodies against NGF receptors. cell line (hybridoma), immunoassay using the monoclonal antibody, and and kits containing these monoclonal antibodies. These cell lines are Clonal antibody I [[G5 producing cell line 1 [IO2, monoclonal antibody ■ Cell line that produces D1 ■D1, monoclonal antibody ■Cell line that produces C8 ■C 8, and the cell line identified as XIFI producing monoclonal antibody XIFI. It will be done.

These four cell lines were listed in the American Type Calculator on December 3, 1990. Char Korekyong, 12301 Burklawn Drive, Rockville, Mary It has been deposited with the State of Land, USA under the following accession number: Cell line 1 [[G5 is A, T , C, C was given accession number CRL10617; cell line ■D1 was A, T, C, C, given accession number CRL10618: Cell line ■C8 is A, T, C , C, accession number CRL10619, and cell line XIFI is A, T, C. , C accession number CRL10620.

The monoclonal antibodies of the present invention can be used to detect the presence of a terminally deleted NGF receptor in a test sample. can be used in a variety of assay systems to determine (if present) Wear. Fragments of these monoclonal antibodies can also be used. The present invention Assays for detecting NGF receptors and/or terminally deleted NGF receptors provide. We found that a distinct NGF receptor similar to that found in rats Developmental control was discovered in human urine. Dystephage and Johnson, Pr. oc, Natl, Acad, Sci, U.S.A., above. during the developmental period No sexual dimorphism was observed during either period, and NGF acceptance in adults No diurnal variation associated with body terminal deletion was observed. Terminal deletion of NGF receptor has a good correlation with the development of functions in peripheral nerves, and secretes NGF receptors. This strongly supports the hypothesis that Schwann cells are the major cell type responsible for this. Sara In addition, NGF receptors in test samples such as biological fluids are associated with abnormalities in peripheral neurons. It can serve as a biochemical marker of development, regeneration and degeneration.

For example, in a first assay embodiment, NGF receptor and terminally deleted neurogenic specifically binds to a specific epitope of the long factor receptor and coated on a solid phase A capture reagent consisting of a first monoclonal antibody or fragment thereof is and terminally deleted NGF receptors, or both. - Contact with the sample to form a mixture. This mixture forms an antigen/antibody complex. Incubate for a time and under conditions sufficient to Then, these complexes with a second monochrome protein specific for the NGF receptor and the terminally deleted nerve growth factor receptor. contact with an indicator reagent consisting of a natural antibody or a fragment thereof coupled to a signal-generating compound. contact to form a second mixture. This second mixture is combined with the antibody/antigen/antibody complex. Incubate for a time and under conditions sufficient to form bodies. Captured on solid phase of NGF receptor and/or terminally deleted nerve growth factor receptor in the tested sample. The presence, if present, of the measurable signal produced by the signal-generating compound Determined by detecting the signal. The amount of NGF receptors present is directly proportional to the signal.

A monoclonal antibody named XIFI was used as a capture antibody, and I[IO2 and Monoclonal antibodies named (compared to XTFI) (recognizing specific and different epitopes of the NGF receptor molecule) were used as indicator reagents. It is preferable to use

In another embodiment, an NGF receptor or terminally truncated NGF bound on a solid support monoclonal antibodies or combinations thereof, or fragments thereof that specifically bind to the receptor; strip, a test sample; and a monoclonal antibody or monoclonal antibody that specifically binds to the NGF receptor. or a combination thereof, or a fragment thereof, with a signal-generating compound attached to the indicator reagent. contact to form a mixture. This mixture is used to form antibody/antigen/antibody complexes. Incubate for a time and under conditions sufficient to ensure that the Test captured on solid phase The presence of NGF receptors in the sample is determined by the signal-generating compound, if present. Determined by detecting the measurable signal generated. N in test sample The amount of GF receptor is directly proportional to the amount of signal generated.

In other assay embodiments, the NGF receptor and/or the terminally truncated NGF receptor The monoclonal of the invention as a competitive probe for detecting antibodies against the body. One or a combination of two or more antibodies, or fragments thereof, are used. For example, NG Coating the F receptor and/or the terminally truncated NGF receptor on a solid phase Can be done. Then, for NGF receptor and/or terminally deleted NGF receptor A test sample suspected of containing antibodies is treated with one of the monoclonal antibodies of the present invention. or a combination of two or more or a fragment thereof with a signal-generating compound attached. Along with the indicator reagent, either the test sample and indicator reagent → solid phase or indicator reagent → solid phase Incubate for a time and under conditions sufficient to form any antigen/antibody/complex. start. The reduction in binding of monoclonal antibodies to the same phase can be measured quantitatively. can be done. NGF receptor and terminally deleted NGF receptor assays confirmed to be negative If there is a measurable decrease in signal compared to the signal obtained from the test sample. contains an anti-NGF receptor antibody and/or an anti-terminal deleted NGF receptor antibody in the test sample. It shows that a body exists.

Although a solid-phase assay has been described, the assays described herein can also be performed without using solid-phase methods. It can also be carried out in solution.

In yet another detection method, immunochemical analysis is performed on fixed or fresh tissue or The present invention can be used to detect NGF receptors and/or terminally deleted NGF receptors in cells. Each monoclonal antibody can be used.

In addition, these monoclonal antibodies can be attached to matrices and used in cell culture. specific NGF receptor protein translocation and terminally deleted NGF receptor protein. Can be used for affinity purification.

These monoclonal antibodies of the present invention or fragments thereof are suitable for NGF receptors and/or or terminally truncated NGF receptors. Main departure The combination of monoclonal antibodies (and fragments thereof) provided by components in a mixture or “cocktail” of anti-NGF receptor antibodies with binding specificities of They can also be used together as minutes.

In all assays described herein, NGF receptor and terminally deleted N Whether a capture reagent or an indicator reagent is used to generate a polyclonal antibody against the GF receptor. It is also conceivable to use it as Polyclonal polyclonals used in these assay embodiments The antibodies or fragments thereof are directed against NGF receptors and/or terminally deleted nerve growth factor receptors. It must be able to specifically bind to the body. Polyclonal anti to use The body may be of mammalian or reptilian origin, and therefore human, goat, horse. Heron or sheep anti-NGF receptor and/or anti-terminal deleted NGF receptor polypeptides. Clonal antibodies can be used. Polyclonal antibodies used in the assay can be used either alone or as a cocktail of polyclonal antibodies. can.

Test samples that can be tested by the method of the present invention described herein include , urine, whole blood, plasma, serum, brain and biological fluids such as cerebrospinal fluid, saliva, sweat, semen, and Examples include conditioned media for cultured human cells. fixed or fresh cells It is also contemplated that tissue and tissues can be used. Solid supports are known to those skilled in the art. The well walls of reaction trays, test tubes, polystyrene beads, and magnetic cubes, nitrocellulose strips, membranes, fine particles (such as latex particles) , sepharose-like beads, and others.

An indicator reagent is a signal capable of producing a measurable signal that can be detected by external means. The label-generating compound (label) is directed against the NGF receptor and/or the terminally deleted NGF receptor. consisting of a substance bound to a specific binding member. In this specification, "specific binding component" is used. "Member" means a member of a specific binding pair. That is, one molecule is part of the second two different molecules that bind specifically to the child by chemical or physical means be. Specific for NGF receptor and/or terminally deleted nerve growth factor receptor In addition to being the antibody member of the binding pair, the indicator reagent also contains biotin and anti-biotin. avidin and biotin, carbohydrates and lectins, complementary nucleotide sequences, receptor molecules and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes F of any specific binding pair, including any habten-antihabten system such as i5. There may be members. Immunoreactive specific binding members include antibodies, antigens, sand NGF receptor and/or terminally deleted NGF receptor as in the switch assay. Antibody/antigen complexes that can bind to the body, bind to capture reagents as in competitive assays antibody/antigen complexes, or co-specific binding compounds as in indirect assays. may be an antibody/antigen complex capable of binding to a member.

Various signal-generating compounds (labels) that can be used include chromogens, enzymes, etc. media, luminescent compounds such as fluorescein and rhodanine, chemiluminescence compounds, radioactive elements, and directly visible labels. As an example of an enzyme are alkaline phosphatase, horseradish peroxidase, β-galactonda - se nato is mentioned. Examples of radioactive elements include 1'I, 31- (and 35 S is raised. It is not important to choose a particular sign, but in itself , or in combination with one or more other substrates to generate a signal. Must be able to do it.

Assays such as monoclonal antibodies of the invention and various wash and assay reagents The reagents used in the test can be provided in kit form, such as vials or bottles. Separate reagents, such as monoclonal antibodies, are placed in one or more containers.

The following examples describe the development, characterization, and development of monoclonal antibodies and assays of the invention. NGF receptors and terminals by describing methods for clinical utility and Figure 2 illustrates the advantages and utility of the present invention for serodiagnosis of truncated NGF receptors.

Example Example 1 Immunology/cell fusion For use as an immunogen, a partially purified preparation of NGF-R1 was prepared as follows. Manufactured. E9b cells were transferred to Chiyao et al.'s 5science 232: 518-521 [Propagated as described in [1986]. Conditionally decane the medium from the cells The solution was adjusted to 0.02% with sodium acetate and stored at 4°C. immunoafinite The preparation of the chromatography resin was described by Axen, R. et al. re 214: 1302-1304 (1967). Identity-purified monoclonal antibody ME20.4 (, 0s (A, H, Ros s) et al., Proc, Natl, Acad, Sci.

USA 81: 6681-6685 (1984)) in a cyanogen bromide activated cell. Phallose-4B (4 mg antigen/ml resin, available from Pharmacia, L This was done by coupling to OCAT■○N). E9b conditional is medium ( 500 ml) was passed onto a ME 20.4-Sepharose column. PB the column Wash with S (20mM phosphate buffered saline, pH 7,4) and wash with acetate buffer (0,2 Elution was with 4M MgCl2 in M, pH 6°5). As a result, Article E9b Specifically, NGF-R1 purified approximately 100 times was obtained from the medium. Column elution The fluid was extensively dialyzed against PBS and concentrated before use for immunization. B at 2 months old ALB/cByJ mice were treated with MPL+TDM Anyuhant (RI B I Immuno Chem Research (RI　B　II　I　mmunochem,　Res, Inc. , ), available from Hamilton, MT), partially purified R1, (approximately 100 the first intraperitoneal injection of 1 μg of total protein) followed by the same amount of protein at 3 weeks. A booster injection containing the substance was given. 10 before first immunization and booster Mice were bled by retroorbital puncture days later. 3 An intravenous injection of approximately 40 μg of total protein was administered 7 weeks after the weekly booster. . After 3 days, the established procedure (Cola and Milneutein, Nature 2 56: 495-497 [1975]: Kohler and Milneyutin, Eur, J. 1mmuno1.6:511-519 [1976], and Bioi (V, T, Oi) and Herzenberg (L, A, ) (erz enberg), Mitsunonyl (B, B, Michelle) and Nigi (S , M. Shiigi) 5 selected Methods in Ce1 lularI mmunology, Freeman (W, H, Freeman) , San Francisco, pp. 351-371 [1980]). 5-1 mouse myeloma cells. Hypoxanthine, aminopterin After selection with and thymidine (HAT), 15% 5% orchid serum (Fe2), Glutamine (2mM), sodium pyruvate (1mM), non-essential amino acids (1mM), 0mM), 2-mercabutoethanol (50JM) and n-(2-hydroxy ethyl) 1 piperazine-N-2-ethanesulfonic acid (HEPES), pH Hybrids were cultured in Dulbecco's modified Eagle's medium supplemented with 7,3 (10mM). held the ma.

Example 2 Affinity labeling of NGF-H using +251-NGF Bocchini (V, Bocchini) and Angeletti (P, U, Angeletti) Proc, Natl, Acad, Sci, USA 64: 787-794 (1969) to purify mouse mandibular NGF and Biochem of varnish (J, J, Marchalonis), J, 113: 177-190 (1969). I understood. Affinity labeling of cell surface and terminally deleted NGF-R species Chi's Proc, Natl, Acad, Sci, USA 83: 4094~ 4098 (1986); sci. 8:231-241 (1988): and Nostephage and Nyoyo. Nson's Proc, Natl, AcadSci, USA 85: 270 -274 (1988) according to the method already described.

Example 3 Radiometric immunosorbent assay (RI SA) Pier 7, (E, A, Pierc e) et al., Anal, Biochem, 153:67-84 (1986). Screen hybridoma supernatants for the presence of antibodies using the RISA test described above. -ning. To explain briefly, IMMULO 2 Removable Well (I mmulo) n2 Removawell Strip (Dynat ech Labs), Alexantria, VA) from PB Goat anti-mouse lgG (50 μl, 50 μg/ml) in S (pH 8,0) and coated overnight at 4°C or for 1 hour at room temperature. Goat anti-mouse IgG After removal, incubate with 1.5% BSA (150 Ig/ml) in PBS for 30 min at room temperature. The interwells were blocked. The wells were washed 3 times with Brideoma supernatant (50 μm) was added and incubated at room temperature for approximately 2 hours.

Wells were washed three times with cold PBS, placed in water, and NG cross-linked to Tsuta251-NGF. Add FR (50 μL approximately 25.000 cpm) to each well and incubate at 4°C for 45 minutes. Incubated. Fill the wells with 0.05% Tween-20R (Sigma Chemical Co., Ltd. , St. Louis, MO) containing four times in cold PBS; Wells were analyzed for the presence of radioactivity.

Example 4 Immunoprecipitation of NGF-R and NGF-Rt Affinity standard with 12'I-NGF The identified receptor-containing sample (50-100μI) was added to the hybridoma supernatant (50-100μI). 100 µm), mouse serum (50 µm 1:1.00 dilution in PBS) or sperm Incubate with monoclonal antibody (5 μm) for 2 to 4 hours at 4°C. Ta. Suspension of goat anti-mouse IgG Sepharose in PBS (10%, v/v ) was added and the mixture was incubated for 1 hour.

Sepharose in tubes containing NGF-R containing 005% Tween-20 Wash twice with PBS (500 μm) while the tube containing NGF-Rt Sepharose inside is 05% serum albumin (BSA), 0.5% sugar and PBS containing 01% Tween-20R. Release the washed resin. Counted for radioactivity or mixed with reduced 5DS-sample buffer. sample loose The solution was incubated with the resin for 1 hour at room temperature and removed by centrifugation. .

Samples were boiled for 90 seconds and applied to a 10% discontinuous 5DS-polyacrylamide gel. Ta. J, Biol, Ch of O'Farrell (P, H, O' Farrell) Em, 250: 4007-4021 (1975) using the buffer system described in Electrophoresis was performed. Using an intensifying screen at -70℃, dry the gel. exposed to a black XAR film.

N after affinity cross-linking to 1251-NGF, immunoprecipitation and gel electrophoresis. Analysis of GF-R or NGF-Rt is performed below by cross-linking immunoprecipitation or CLIP assay. It is called Sei.

Example 5 Radiolabeling of monoclonal antibodies Bolton (A, E, Bolton) and Hunter (W, M, Hunt) er) method (Polton and Hunter, Biochem, J, 133: 5 29-539 [1973 Co. L), affinity purified antibody (50μ g) in 12J-Voltrane 1 Interreagent (2200Ci/mmol, 1mC1 ) was radioactively labeled. Contains 0.2% gelatin and 0401% N a N 3 E conopac 1-DG cells equilibrated in PBS. Taromadock on Rum (Violad Labda, Rich St., California) By subjecting the '25I-labeled antibody to unreacted 1251-port separated from the N-Hunter reagent.

Example 6 immunoblotting 2% n-octylgluco/t, 0.65M NaCl, 1mM PMSF and 1 mM iodoacetamide 4) was used to solubilize NGF-R from E9b cells. The solubilized preparation was Mix with SDS-sample containing % β-mercaptoethanol and boil for 90 seconds. Alternatively, use LX directly without boiling by mixing with non-reducing SDS-sample buffer. .

Samples were separated on a 10% discontinuous SDS-polyacrylamide gel. Front side dyed Molecular weight markers ()\iorad labda, lysothymondo, calipho (available from Lunia) was applied in parallel lanes. Towb (H. Towb) in) et al.'s Proc. Natl. Acad. Sci. USA 7 6: 4 350-4 354 (1.9 79) l As described above, protein The samples were electrophoretically transferred to nitrocellulose membranes. PB containing 5% skimmed dry milk Incubate the membranes for 1 h at room temperature in S. horsetail) containing 0.05% nonfat dry milk. Rinse once with PBS and add affinity-purified monoclonal antibodies ( 25 μg/ml) for 2 hours. 05% skimmed dry milk and The membrane was rinsed three times for 5 minutes each in PBS containing 0.05% Tween-20.

The membranes were then incubated for 2 hours in the presence of 1251-labeled goat anti-mouse IgG. I bet. Radioactively labeled antibodies were added to 0.5% nonfat dry milk and 0.05% Tweet. diluted in PBS (3 x 10' cpm/ml) containing 20% Diluted once by myself. 1. Dry the film using an intensifying screen at -70°C. The film was exposed to an exposed XAR film until the film became sensitive.

Example 7 Antibody competition studies Affinity-purified antibodies are radiolabeled with 1251-Porton-Hunter reagent. did. Cells (E9b or Ltk-) were solubilized as above. E9b thin Cell conditioning can be done using NCR-Rt directly in the culture medium or by using human NCR-R. Immunoaffinity chroma on resin constructed using antibody (ME20.4) Purified by chromatography. The column was washed with PBS, 20mM sodium phosphate. Mu buffer (containing 0.55M NaCI, pH 7.4), PBS, 50mM CA Washed sequentially with PS buffer (pl-19.8). NGR-Rt is CPAS buffered The column was eluted using a solution (pH 11.5). IM HEPES buffer (p The column fraction was brought to pH 7.4 by adding H7.0).

Immuron 2 remover was added with a selected monoclonal antibody of the present invention (5 ml) in PBS. 0 μg/ml) (5077+) overnight at 4°C. 1 in PBS .

Other binding sites were removed by adding 5% BSA (150 μl) for 3 minutes at room temperature. I blocked the position. Wells were washed three times with cold PBS, followed by receptor-containing preparation. (50 μl) and incubated for 90 minutes at room temperature. Cool the wells Washed 3 times with BS, and then treated with a designated 125I-labeled monoclonal antibody (2.5~ 5x10'cpm/well) and incubate for 45 minutes at 4°C. I started.

Then, the wells were washed 4 times with PBS containing 0.05% Tween-20 and released. I counted the radiation.

Example 8 General procedure Bradford CM. using crystalline BSA as standard. M. B rad Anal. Biochem. 72: 248-3454 (19 Protein concentration was determined by the method described in 76). LKB Ultro Scan (UltroScan) Perform laser density measurement using an XL laser density meter. It was. Protein A-Sepharose Monoclonal Antibody Purification System (Biola Hybridoma supernatant Antibodies were affinity purified from the solution. Boehringer Mannheim (India) ScreenType (Napolis, Indiana) )” kit was used to determine the isotype of the antibody.

Example 9 Production of monoclonal antibodies Monoclonal antibodies were produced against human recombinant NGF-Rt as follows. I let it happen. Briefly, the E9b cell line (5science 2 by Chao et al. 32518-521 [11986]), transfected with human genomic DNA. Mouse L cells expressing the NGF receptor gene were maintained in a culture medium for 3 to 4 days. NGF-R1 was recovered from the conditioned medium. Is the conditionality of E9b cells dependent on the medium? The NGF-R1 obtained from NGF-R1 was used as a receptor source for immunization. E9b conditional is cultured The NGF-Rt obtained from the ground is the same as that previously reported elsewhere. To demonstrate this, E9b conditioning involves affinizing receptors in the culture medium with Tea-purified, immunoprecipitated with antibody ME 20.4, and 5DS-polyacrylamide Separated on gel. As a result, as shown in Figure 1, the predicted apparent molecular weight 6 Sakuraji protein of 6,000 daltons was observed. NGF-R1 under E9b conditions The following was immunopurified from the culture medium and used to immunize BALB/cByJ mice. Department Serum from mice given primary immunization and booster injection of fractionally purified NGF-Rt detected antibodies against intact NGF-Rj by RTSA and immunoprecipitation assays. It was positive for activity. The lung cells of this mouse were used as N5-1 mouse myeloma. fused with cells.

Hybridomas secreting antibodies against NGF-R were first grown as described in Example 3. It was identified using a RISA assay. More than twice the bank ground / Wells (34/1056 wells) showing GNAR were rescreened by immunoprecipitation. This assay showed that 18 out of 34 wells had no antibody against NGF-R. It was found to be positive for the presence of Screening after cell line expansion Five cell lines remained positive for antibodies to NGF-R, limiting these Cloning was performed by dilution. After cloning and expansion, 4 hyperdos The M. ma strain remained positive for antibodies to NGF-R. These hybrids Cell line 1 producing monoclonal antibody I [[05, monoclonal Null antibody ■Cell line that produces D1, monoclonal antibody ■D1, produces monoclonal antibody ■C8 Cell line ■C8, and cell line XIFI that produces monoclonal antibody XIFI. and identified it. These monoclonal antibodies were tested for class and As characterized using subclass-specific anti-mouse immunoglobulin antisera, All were shown to be IgG+, i antibodies.

Example 10 Analysis of antibodies using CLIP For the ability to immunoprecipitate +2'I-NGF-affinity-labeled receptors. Clonal antibodies were tested. Samples were separated on a 5DS-polyacrylamide gel. The molecular identity of the immunoprecipitated species was confirmed. All four monoclonals of the invention by antibodies (designated as 111G5, ■D1, ■C8, and XIFI) The results of immunoprecipitation of affinity-labeled receptors from E9b cells are shown in Figure 2. At the same time, 90.000 Mr species appeared on the gel autotraphogram. NGF stuff subtracting the mer (13,000 Daltons), approximately so for cell surface receptors; oo.

This is the net molecular weight of Dalton. Monochrome against NGF-R in human melanoma cells The same protein was immunoprecipitated using the null antibody ME20.4 (Ross et al. Proc, Natl, Acad, Sci, USA 81: 6681-66 85 (1984)), control mouse IgG, i antibody (MOPC21 ) or using a monoclonal antibody (Ab192) specific for rat NGF-R. affinity-purified receptors were not immunoprecipitated when -(C, E, Chandler) et al., J. Biol, Chem, 259: 6882-6889 [1984]). The four monoclonal antibodies produced A preparation containing affinity-labeled NGF-R1 was prepared as shown in Figure 3. Immunoprecipitating a protein with an apparent molecular weight of 63,000 daltons from was completed. Similarly, subtracting the monomer of NGF, the acceptance of the truncated form A net molecular weight of approximately 50,000 Daltons is obtained for the first. 12'I-N GF-NGF-R+ complexes were not immunoprecipitated by antibody MOPC21.

Determine whether the above two antibodies recognize both high-affinity and low-affinity NGF-R. To determine the affinity-labeled NGF-R solubilized from 5H-3Y5Y cells, The CLIP assay was performed as described in Example 4 using 5H-8Y5 Y cells possess clonal affinitized forms of the NGF-receptor. y form) (K, H, 5onnenfeld) and Inii (D, N, I 5hii), J, Neurosci, 5: ] 717-] 728 [See 1985). As shown in FIG. One all monoclonal antibody as well as antibody ME20.4 is 5H-8Y5Y Immunoprecipitation of 90.000 Mr NGF-R”I-NGF-R complex from cell receptors It rained. No labeled substance was immunoprecipitated by MOPC21.

Example 11 Species cross-reactivity All monoclonal antibodies of the invention were tested using the two-site RISA described in Example 3. It bound to NGF-R1 from monkey urine. However, the chick embryonic dorsal root ganglion, Antibodies did not bind to NGF-R from rat supraneuroid ganglia or PCl.2 cells. Here it is.

Example 12 Immunoplot of cell surface NGF-R To further characterize the specificity of antibody binding, NGF- Immunobronting studies were performed using R. Receptor sample in the absence of reducing agent If prepared and not boiled before separation on the Kel, 2 Two monoclonal antibodies (D1 and XIFI) have approximately 68,000 daltons. Combined with major protein right of molecular weight. The control antibody MOPC21 had no effect. No munoblotting occurred. Further experiments revealed that it was long enough. Upon film exposure, all antibodies of the present invention were able to immunoblot NGF-R. It was shown that the However, the obtained autoradiographic signal The intensity was always lower for monoclonal antibodies ■G5 and ■c8. acceptance Body samples were exposed to a reducing agent (β-mercaptoethanol) and boiled prior to electrophoresis. When the cells were incubated, no antibody binding occurred, as shown in Figure 5B.

Example 13 Selection of monoclonal antibodies for epitope mapping and assays The present invention determine whether a monoclonal antibody binds to a specific receptor epitope Therefore, a solid-phase competition study was conducted. All monoclonal antibodies were tested on solid phase; It was also used as a radioactively labeled (top) monoclonal antibody. In this assay, Retention of the radiolabel in the well allows the top and bottom antibodies to move onto the receptor protein. was shown to recognize distinct epitopes of . The only exception to this is This occurred when a single antibody bound to repeated epitopes on the container. competitive research The results are as shown in Figures 6A-D. Antibody II [G5 is monoclonal antibody ■D1 , ■ C8, XIFI and the previously developed monoclonal antibody ME204. bound to an epitope on NGF-Rt that is distinct from the epitope recognized by It clearly showed that. Probe each monoclonal antibody against itself In this case, retention of radioactivity in the wells can be achieved using control antibody MOPC21. It was no bigger than if it had been. These results demonstrate that the monoclonal antibody of the present invention showed that it does not recognize repeated epitopes on the receptor.

Distinct sites on the receptor can be isolated for use in the development of two-site radiometric immunosorbent assays. Two recognizing monoclonal antibodies were selected. Based on the results of the above competitive research, Antibodies IG5 and XIFI were selected. NGF-R (shown in Figure 7A) or NG When assaying F-R1 (shown in Figure 7B), radiolabeled I[[G5 and anchor - (capture) antibody in combination with XIFI to achieve the highest specific counts per well. Gave a big hold. To assess non-specific binding, E9b cells and E9b cell lines were Conditional assay of Ltk-cells or Ltk-cell conditional assay of media in parallel. did.

These studies indicate that nonspecific binding in the assay is conditionally soluble compared to medium. This result was shown to be higher when using cultured cell preparations. Anchor MOPC21 ( Even when used as a capture antibody, nonspecific binding could be well evaluated.

To test the primary nature of the assay, it had been concentrated by ammonium sulfate precipitation. Serial dilutions of NGF-R1 were tested. As shown in Figure 8A, less per well The amount of NGF-Rt added and retained per well up to 30.000 cpm. A first-order correlation was observed between the amount of 125T and G5. Example 3 The data obtained from the two-site RISA were analyzed using the CLIP assay of Example 4. The data obtained were compared. Figure 8 shows the results using two-site RISA and CLIP assays. The results show a close correlation (R-0,998) between the relative values obtained.

Example 14 Sample collection and preparation Urine samples were collected from 70 normal human subjects aged 1 month to 68 years. Third Urine from four pregnant women (33 to 41 years old) in their third month of pregnancy was also collected. polypropy Ren sample container (Scientific Products) Collect urine in The cells were immediately placed on ice and frozen at -80°C within 2 hours of collection. from collection Urine samples were routinely assayed within two weeks. The sample was heated to -80°C at least Even after 3 weeks of freezing, NGF-Rt or creatinine assays are less effective than fresh urine. There was no decrease in the i value. For newborns, use cloth diapers or U-hang (Available from Ho]l1ster, Kirkspill, Missouri) Urine was collected using

For assay, samples were thawed at 4°C, centrifuged at 13,000×g for 5 min, Dilute to 11 with 0.51vl HEPES buffer (pH 7,0), then add the following: into the assay plate. Assay bra used as negative control The solution was 20mM phosphoric acid, 160mM NaC] (phosphate buffered saline [PBS 1) (diluted to 11 in HEPES buffer).

Example 15 creatinine and protein Tietz (N, W.) for thawed and centrifuged urine samples. Textbook of C11nical Chemistry, Saunder (W, B, 5unders Co, ), Philadelphia, 1278 Creatinine was assayed using the picric acid method described on page 1280 (1986). I did it. Proteins were measured using blood serum albumin as a standard. Anal, Biochem, 72: 248-254 (1976). Determined in urine samples by the method described.

Example 16 2-site RISA Monoclonal antibodies of the present invention that bind to specific epitopes on receptor proteins Nerve growth factor receptor (NGF-R) and terminally deleted nerve growth factor receptor Developed a two-site radiometric immunosorbent assay (RISA) for (NGF-Rt) did. XIFI was chosen as the anchor (capture) antibody and It was used to bind NGF-R or NGF-Rt to the solid phase. Presence of NGF receptor 1251-mG5 in the final incubation step to detect the presence of 1251-mG5. (5XIQ6cpm/well) was used as the indicator reagent.

The assay of the present invention is described in Pierce et al., Anal, Biochem, 153: 67-74 (1986). . Monoclonal antibody XTFI was added to 50 μg/ml in PBS (pH 8,0). Dilute and apply Imron Remover Well Strip (Dynatic, Chante) (available from Co., Ltd.) in a volume of 50 μm and incubated overnight at 4°C. I was incubated. Fill the unreacted area on the well with 1.5% serum albumin for 1 hour. Dynamic plate washer 1, available from Dynatic, Chantilly, Bernier) Washed with S (pH 8,0). Diluted urine sample (50 μl) as described in Example 14 were added in four portions for 1 hour, followed by washing. Lactoperoxidase method (Marcha Ronis, Bjochem, 'J, 113299-305 [1969] ), monoclonal antibody mG5 at 4000-5000 cpm/fmol It was iodinated to a specific radioactivity of e. ”I-I [[05 (500,000 cpm) each 0.1% Tween 208 (Ngma Keke) was added to the wells for approximately 45 minutes on ice. 4 times with ice APBS containing (available from Michal, St. Louis, MO) Washed. Separate the wells, place them in tubes, and place them in a Beckman comma counter. (Model 5500) for 1 minute. Purification with end deletion count 7 minutes Always normalized to a standard sample of recombinant human receptor protein. The data is μ as nanograms of NGF-Rt per g creatinine or urine protein Expressed as nanograms per mg. In some experiments, the anchor to XIFI +251-I can be saturated with urine NGF-Rt [IG5 binding] I[[G5 affinity for GF-Rt was evaluated. for these studies , I [105 was iodinated to a specific activity of 600-800 cpm/fmo I, and the above Binding was performed for 45 minutes with various concentrations of labeled I[[G5. K, and B.5. Values were determined by scratch yard plot. B, , , is μg cre Expressed as nanograms (ng) 1-NGF bound per atinine, carried out Determined as described in Example 15.

Example 17 Affinity labeling/crosslinking immunoprecipitation of NGF-Rt (CLIP) Nostefa George and Johnson's Proc, Natl, Acad, S+j, U S A Urine samples of subjects of various ages ( Example 14) NGF-Rt in 1251-NGF (specific activity = 2000-25 00 cpm/fmol). Monoclonal antibody ME2 Immunoprecipitation of NGF-NGF-R1 complexes was performed using 0.4 or XIFI. Ta. Samples were transferred to 5DS-PAGE and Kodak X-0-MAT1' film (Kodak For autorunography using a solenoid (Rochester, New York), Proc of Nostephage and Njonson, Natl, Acad, Sc i, U.S.A. Treated in the same manner as described in the above document. gel/autoradiog The marks that appear on the ram are measured using an LKB Ultrascan XL laser densitometer (LKB , Piscataway, New Chassis).

Example 18 statistics Urine N obtained from various groups according to the procedure described in Examples 16 and 7. Statistical differences in GF-Rt values were determined by one-way ANOVA. and subsequent Newman-Keuls' It was determined using analysis.

Significant differences were determined at 0.0 lH. 1251-NGF binding to NGF-Rt The binding constants of were compared using Student's t-test.

The two-site RISA described in Example 16 was used to detect NGF-Rt in human biological fluids. It was found to be a rapid and reliable means of quantifying Shown in Figure 9A As such, the assay is 50. OOOcpm, purified recombinant receptor with terminal deletion was linear with respect to the amount of For E9b conditional, the internal standard of the medium is 10% as usual. ．． 000 cpm. As shown in Figure 9B, the assay can also be performed with a 25 μm volume or The amount of urine added (diluted to 11 in HEPES buffer) was Met. The sample-blank ratio varies from 31 to 30 depending on the content of NGF-Rt in the sample. It was in the range of 301. The accuracy of the analysis was determined using urine from 17 subjects. 2-8%, with inter-assay variation of less than 4%. In addition, in adult urine NGF-Rt was found to change by only 5% over a period of six months. N GF-Rt protein is very stable in urine, lasting at least 2 months at -80°C. It was found that it could be stored for a long time without losing its activity. In addition, the urine sample was kept at room temperature for 8 Can be left for some time without compromising NGF-R1 activity or creatinine levels It was decided that. However, boiling a urine sample reduces creatinine levels. Activity was lost without any effect. Current status of the assay described in Example 16 The throughput is such that a person can assay 65 samples in 4 batches per day. Ru.

Absolute concentration of urine NGF-Rt obtained in Example 16 as a function of age There was a slight tendency for levels to decrease with age. urine NGF The absolute amount of -Rt ranged from 56 to 1200 ng/ml urine. Shown in Figure 10 As a function of age, expressed as ng NGF-Rt/μg creatinine, A clear and dramatic urinary NGF-Rt regulation was observed. Age of 1 month (from what I looked into) NGF-Rt levels are very high in the earliest years of life and decline rapidly during the first year of life. It took a while. The decline in NGF-Rt levels is more modest for ages 1 to 15 years. He reached the adult level at the age of 15. For those aged 15 to 68, the age of 1 month is It remains essentially constant as 5% of the average value obtained from the test subjects. 70 assayed Among normal human subjects, only - human subjects fell outside the curve created and shown in Figure 10. It was When this subject was re-assayed 6 months later, it was found that the test result was within the normal range. It was found that it is included in Therefore, the reliability of the assay is >98% . The insert in Figure 10 provides an assessment of the decrease in urinary NGF-Rt during the very early developmental period. An expanded X-axis plot of the data obtained is shown. First month to 6 months of life During this period, urinary NGF-R1 rapidly decreased in the urine. During the developmental period from 0.5 years old to 35 years old However, the decline in urinary N during the first four years of life is apparently more modest. This suggested that the decrease in GF-Rt was bimodal.

NGF-Rt is secreted in the urine and is a large protein, so urinary NGF-Rt R1 was also expressed as ng/mg urine protein. Figure 11 shows the relationship between a newborn and an adult. Expressed per mg protein, except that the difference between Figure 2 shows similar rules for the development of NGF-R1 when In addition, mg Data when expressed per protein compared to when normalized to creatinine The fluctuations were much larger. However, how did you normalize the data? Regardless, a similar developmental pattern is evident in human urinary NGF-Rt. there were.

To statistically evaluate changes in urinary NGF-R1 as a function of age and sex, Fig. Put the 10 data into age bins and calculate the average value of these groups. compared. In addition, we compared the NGF-Rt levels observed in pregnant women with those in normal adults. compared.

These data are shown in Table 1.

Table 1 Analysis of urine NGF-Rt value Age (years old) * NGF-Rt (ng/μg creatinine) Number of observations 00 8~0.10 7.00±2.36** 53~3.5 0.91±0.23* *515~68 (Pool end ll+F) 0.31±0.06 3130~5 0M O, 29±0.06 1415~68F 0.34+0.06 1733 ~41 (pregnant woman) 0.58±0.09** 4* Obtained from various age ranges Values were pooled and expressed as mean±SD. The number observed in each group is shown on the far right. Ta. M, male, F female. Urine was collected from women who were 75 to 85 months pregnant.

**p<0.01 synthesized by E9b cells compared to all other groups production of mono-intellectual antibodies against the soluble, truncated form of NGF-H I let it happen. NGF-Rt obtained from E9b cells differs in molecular weight and unlabeled NGF. Regarding displacement of 1251-NGF binding, cultured Schwann cells were conditioned with medium ( Distephage and Johnson, Proc, Natl.

Acad, 3ci, USA 85: 270-274 [1988 co) and Conditioning of melanoma cells is carried out using medium (Zupan, A. et al., J. Bio. Chem, 264:11714-11720 [1,989]). The results were similar to those of NGF-RI. The four monoclonal antibodies of the present invention are We immunoprecipitated protein-labeled receptor species to varying degrees, but all of these Clonal antibodies target whole (cell surface) or truncated forms of NGF-R. It is obvious to recognize. Furthermore, all of these antibodies are 5H-3Y5Y human It binds with high affinity to NGF-R localized on the cell surface of neuroblastoma cells. This thin cell lines are reported to express the most high-affinity NGF-R (Sonnen et al. Felt and Ishii, J, Neurosci, 5:1717-1728 (1985)). The monoclonal antibody of the present invention has a truncated low affinity receptor. The fact that it recognizes a high-affinity receptor produced in response to a low-affinity receptor High-affinity and high-affinity NGF-Rs have overlapping regions of sequence homolon- or the same protein.

core (low affinity) by binding of regulatory proteins located within the plasma membrane Evidence suggests that high affinity is conferred on NGF-R proteins (and For example, Hosang (M) and Shooter (E, M, 5hoo) ter), J, Bjol.

Chem, 260: 655-662 [1985]; Green (sH, G reen) and Greene (L, A, Greene), J, Biol, Che m, 26] +15316-15326 [], 986] 1 and hempste Sodo (B, L, Hempstead) et al., 5science 243373-3 75 [1989]).

The monoclonal antibodies of the present invention produced in this study were tested by both in-phase and immunoprecipitation. In the assay, ``1-NGF-affinity-labeled particles were shown to bind to human NGF-R.'' Screening was based on competency. All monoclonal antibodies of the present invention are It cross-reacted with the NGF receptor that had been deleted at the end of the protein, but the There was no cross-reaction with. Therefore, the monoclonal antibody of the present invention It specifically binds to growth factor receptors and terminally deleted nerve growth factor receptors, and It also specifically binds to monkey terminally deleted nerve growth factor receptors; Does not bind significantly to chicken nerve growth factor receptors. against H1-NGF-R There are two other reports regarding the origin of the monoclonal antibody produced by the monoclonal antibody, but both are new. 1) or cross-react with receptors from rats (Ross et al., Proc. t] Acad, Sci SA 81: 6681-6685 [1984 Co. and NMarano et al., J. Ncurochem, 48:22. 5-232 [1987]). NGF- from humans, rats and chickens Comparison of R cDNAs reveals that the encoded receptor amino acid sequences are highly conserved. (e.g., Ngyoson et al., Ce1147:51I5-554 [1986 ]: Radeke (M, J, Radeke) et al., Nature 325:59 3-597 [1987]: x Nfors (P, E nfors) et al., N euron], : 983-996 [1988 Co., Niskanton (E), E5candon) and Chao, Dev, Brain Res, 47: 18’7”196 [1989]; and Large (T, H.

1, arge) et al., Neuron 2:1123-1134 [1989]. (see). This explains why it is difficult to produce antibodies that cross-react with various species. It may explain what happened.

The immunoplot experiments described herein were performed using the reduced form after transfer to nitrocellulose. It was shown that the monoclonal antibody of the present invention does not bind to NGF-R. this This means that these antibodies recognize NGF-R species in a conformation-dependent manner. This suggests that The present inventors demonstrated that these antibodies interact with receptors expressed in bacterial systems. It was determined in a separate experiment that Glob (P, MG) rab) et al. J, Biol, Chem, 260: 8044-8049 (1 As described in 985), these antibodies rather than recognizing core proteins.

Four novel monoclonals of the present invention that bind to specific epitopes on NGF-R This antibody can be used to measure NGF receptors or terminally deleted nNGF receptors in a large number of samples. This makes it possible to develop highly sensitive assays of the invention that can be used to

The assay of the invention can also be used to measure total NGF-R extracted from various sources. You can also be there. Measurement of NGF receptor using two-site RISA was previously described. provides further advantages over the conventional method. Because the NGF receptor is a ligand This is because it can be assayed in the absence of (NGF). 2 parts RISA Similar results were obtained when measuring NGF-R1 using the and CLIP assays. Ta. 2Fa-position RISA also regulates NC;F-Rt in human urine during development. It was also used to investigate the emissions caused by Antibodies that bind to different receptor epitopes are , confirming the specificity of antibody binding to the receptor in tissue sections using immunohistochemical methods provides important tools that can be used to Recombinant receptor overexpression In combination, these novel monoclonal antibodies also facilitate the study of NGF-R structure. It can also be used to develop immunoaffinity purification methods for Nisuda.

Other modifications and variations of the specific embodiments of the invention described herein will be apparent to those skilled in the art. It will be clear. Accordingly, it is intended that the invention be limited according to the scope of the appended claims. This is for illustration purposes only.

FIG. 1 FIG.2 lane FIG.3 FIG. 6A MOFC21Ma2O, 4mG5 wDI - C8XIFI, it's not true! , Book of books MOPC21Ma2O, 4IIIG5 'MDI N Ro] Go CB > Go F1 Gu Kasei Nephew Akitai FIG, 6C MOPC21Ma2O, 4lllG5 'nDl ■C8XIFI fixed, Do f%, *hairy book.

FIG, 6D MOPC21Ma2O, 4lllG5 '5ZIIDI vcho KB XI Nippon Hiki 11-Pyohama Mobile Body FIG. 7A Fixed egg challenge 1 vinegar version FIG. 7B FIG, 8A CLIP (lol) FIG. 8B FIG.9A rhNGF-Rt, ng FIG.9B ゛　J- Otori 9 L NGF-Ri, ng/ug glare +=n NGF-R↑, nq/m9 15n/ <’7’f International Search Report Continuation of front page (51) Int, ci, 5 Identification symbol Internal reference number GOIN 3315 3D 8310-2J331577B 9015-2J //(C12P 21108 C12R1:91) I

Claims (16)

[Claims] 1. Specific for human nerve growth factor receptor and terminally deleted human nerve growth factor receptor It also binds specifically to the terminally deleted monkey nerve growth factor receptor, and Monoclonal antibodies that do not significantly bind to the Watori nerve growth factor receptor or piece. 2. Hybridoma A. T. C. C. Accession number CRL10617, hybrid Ma A. T. C. C. Accession number CRL10618, hybridoma A. T. C. C ．． Accession number CRL10619 and hybridoma A. T. C. C. Accession number C Monochrome obtained from a hybridoma selected from the group consisting of RL10620 2. The monoclonal antibody according to claim 1, which has the binding specificity of a null antibody. 3. Specific for human nerve growth factor receptor and terminally deleted human nerve growth factor receptor It also binds specifically to the terminally deleted monkey nerve growth factor receptor, and Monoclonal antibodies that do not significantly bind to the Watori nerve growth factor receptor or Hybridoma that produces fragments. 4. Hybridoma A. T. C. C. Accession number CRL10617, hybrid Ma A. T. C. C. Accession number CRL10618, hybridoma A. T. C. C ．． Accession number CRL10619 and hybridoma A. T. C. C. Accession number C Claims having the identifying characteristics of a hybridoma selected from the group consisting of RL10620 The hybridoma according to item 3. 5. A. T. C. C. Deposited with A. T. C. C. Accession number or CRL10617 A monochrome gene identified as IIIG5 produced by a hybridoma null antibody. 6. A. T. C. C. Deposited with A. T. C. C. Accession number or CRL10618 A monochrome gene identified as VIID1 produced by a hybridoma null antibody. 7. A. T. C. C. Deposited with A. T. C. C. Accession number is CRL10619 The monoclonal protein identified as VIIIC8 produced by the hybridoma local antibodies. 8. A. T. C. C. Deposited with A. T. C. C. Accession number is CRL10620 A monoclonal clone identified as XIF1 produced by a hybridoma null antibody. 9. Contains human nerve growth factor receptor or terminally deleted human nerve growth factor receptor There may be a human nerve growth factor receptor in the test sample and/or a terminally deleted human 1. A method for determining the presence of nerve growth factor receptor, comprising: 1a. bound to solid phase a monoclonal antibody or a fragment thereof, wherein the antibody is a human nerve growth factor receptor. specifically binds to the human nerve growth factor receptor and the terminally deleted human nerve growth factor receptor. It also specifically binds to the nerve growth factor receptor in rats or chickens. Forming a mixture by contacting the test sample with something that does not bind significantly to the body death; b. The mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes. cuveate; c. The complex was synthesized into human nerve growth factor receptor and terminally deleted human nerve growth factor receptor. It specifically binds to the human body and also specifically binds to the terminally deleted monkey nerve growth factor receptor. A second monochrome that does not bind significantly to the chicken or chicken nerve growth factor receptor. signal generation capable of producing a measurable and detectable signal bound to a null antibody; contacting an indicator reagent consisting of a compound to form a second mixture; d. said second mixture Incubate the substance for a time and under conditions sufficient to form antibody/antigen/antibody complexes. Bate; e. detecting the human pathogen in the test sample by detecting the measurable signal generated. Determining the presence of neural growth factor receptors or terminally deleted human nerve growth factor receptors A method characterized by: 10. The signal generating compound is a luminescent compound, a chemiluminescent compound, an enzyme. and a radioactive element. 11. The monoclonal antibody or fragment thereof of step (a) or (c) is about 5 Specific for epitopes on molecules of 0,000 Daltons or approximately 80,000 Daltons 10. The method of claim 9, wherein: 12. the monoclonal antibody or fragment thereof bound to the solid phase of step (a); or The monoclonal antibody or fragment thereof bound to the indicator reagent in step (c) is Ibridoma A. T. C. C. Accession number CRL10617, hybridoma A. T. C. C. Accession number CRL10618, hybridoma A, T. C. C. Entrustment No. CRL10619 and hybridoma A. T. C. C. Accession number CRL1 A monoclonal clone produced by a hybridoma selected from the group consisting of 0620 10. The method of claim 9, wherein the method has the binding specificity of an antibody. 13. The monoclonal antibody or fragment thereof of step (a) comprises A. T. C. C. Deposited with A. T. C. C. Hybridoma with accession number CRL10620 It is identified as an XIF1 monoclonal antibody obtained from a cell line, The monoclonal antibody according to claim 9. 14. The monoclonal antibody or fragment thereof in step (c) is a compound of A, T. C. C. Deposited with A. T. C. C. Hybridoma with accession number CRL10617 It is identified as a IIIG5 monoclonal antibody obtained from a cell line. 10. The monoclonal antibody according to claim 9. 15. human nerve growth factor receptor or terminally deleted human nerve growth factor receptor in the test sample. An assay kit for determining the presence and/or amount of a container comprising: a. Hi specifically binds to human nerve growth factor receptor and terminally deleted human nerve growth factor receptor. , which also specifically binds to the terminally deleted monkey nerve growth factor receptor, in rats or chickens. Monoclonal antibodies or fragments thereof that do not significantly bind to nerve growth factor receptors a capture reagent consisting of; b. Specific for human nerve growth factor receptor and terminally deleted human nerve growth factor receptor It also binds specifically to the terminally deleted monkey nerve growth factor receptor, and Monoclonal antibodies that do not significantly bind to the Watori nerve growth factor receptor or characterized in that it consists of an indicator reagent consisting of a signal-generating compound bound to the fragment. kit. 16. The monoclonal antibodies of (a) and (b) are obtained from hybridoma A. T. C. C. Accession number CRL10617, hybridoma A. T. C. C. Accession number CRL10618, hybridoma A. T. C. C. Accession number CRL10619 and hybridoma A. T. C. C. Is it the group consisting of accession number CRL10620? The binding specificity of monoclonal antibodies produced by hybridomas selected from The assay kit according to claim 15, comprising: