CA2097309A1

CA2097309A1 - Immunoassay and monoclonal antibodies useful for detecting truncated nerve growth factor receptor

Info

Publication number: CA2097309A1
Application number: CA002097309A
Authority: CA
Inventors: Peter S. Distefano; Margaret Clagett-Dame
Original assignee: Individual
Current assignee: Abbott Laboratories
Priority date: 1990-11-30
Filing date: 1991-12-02
Publication date: 1992-05-31
Also published as: AU9164991A; WO1992009631A1; EP0559834A1; JPH06503722A; EP0559834A4

Abstract

Monoclonal antibodies which specifically bind to human nerve growth factor receptor, human truncated nerve growth factor receptor, monkey truncated nerve growth factor receptor, and which do not significantly bind to rat or chick nerve growth factor receptor, and hybridomas which produce these monoclonal antibodies. Also provided are assays which utilize these monoclonal antibodies to determine the presence of human nerve growth factor receptor or human truncated nerve growth factor receptor in a test sample, and asssay kits containing these monoclonal antibodies.

Description

~0 92/Og631 PCr/US91/08g6~ ., IMMUNOASSAY AND MONOCLONAL ANTIBODIES USEFUL FOR DErECTING
TRUNCATED NERVE GROWrH FACTOR RECEPTOR

~ckpround of the Inven1ion This invention relates generally to monoclonal antibodies which bind to nerve growth factor receptor, and more particularly, relates to an immunoassay and 1 0 monoclonal an~ibodies useful for ~etermining the presence of human nerve growlh factor receptor or human truncated nerve growth factor receptor in test samples.
Nerve growth factor (NGF) receptors have been characterized on neuronal cell lypes and have been categorized into high and low affinity forms of the receptor.
1 5 it ~raditionally has been thoughl that the high affinity form of the receptor m~diates the trophic and tropic actions of NGF in neurons, while the significance of the low affinity forms has remained uncertain. The low affinity forrn of the NGF raceptor aiso has been identified on non^neuronal cells in tissue culture prr;~parations. A. Z. :
Zimmerman and A. Sutter, ~m~2Q,L 2:879-885 (1983); S. T. Carbonetto and R. W.
Stach, Dev. ~rain~, 3:463-473 (1982). Defined cultures of highly purified Schwann cells have been used to demonstrate the presence of low affinily NGF binding sites on the S~!hwann cell surface. P. S. DiStefano and E. M. Johnson, J Neurosci.
8:231-24-i (1988); J. G. Assouline and J. N. Pantazis, Exp. Cell Res. 182:499-51-512 (1989). ~ Q. NGF binding sites appear on rat Schwann cells in 2 5 response to axotomy. M. Taniuch~ L, PrQc. Na~L. Aca~5ci. U~ 83:4094-4098 (1g863. in addition, during early development when the axon-Schwann cell contactis immature, high levels of NGF receptor are localized in peripheral nerve, suggesting that disruption of axonal-Schwann cell contact results In high levels of NGF receptor expression on the Schwann ceil surface. It has been postulated that low 3 0 affinity NGF receptors are reguiated in Schwann cells by physical contact with an element on the axon, such that innervation from ~eveloping or regenerating axonsrepresses the NGF receptor protein (see, for example, ~i. Taniuchi ~, ~ and P.
S. DlStefanG and and D. M. Chelsea, Soc. Neurosci. Abs~ 14:1268 ~1988]) and its mRNA (G. Lemke and M. Chao, D~Yqlopment 102:499-504 ~1988]).
3 5 ~-We rer,ently showed that Schwann cells maintained in tissue c~lture shed a truncateri form of the NGF receptor (NGF-Rt) into their medium..~ P. S. DiStefano and E. M. Johnson, Proc. Natl. Acad. Sci USA 85:270-274 (1g88). Several NGF
receptor-bearing celis shed NGF-Rt into their media, including rat 4 0 pheochromocytoma (PC12) (P. S. DiStefano and ~. M. Joh~son, Proc. Natl. ~d~ci , .
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wo 92/09631 PCr/VS9l/08~68 ~

. ~iO. rat schwannoma (P. S. DiStefano and E. M. Johnson, IkO and human myeloma (A875) (A. A. Zupan ~LaL, J~ BjQI~ ~h~m~264:1t714-11720 (19B9), although not to the extent of Schwann cells. Interestingly, NGF-dependent neurons of J
the superior cervical ganglion shed only a high molecular weight or whole form of 5the receptor. (P. S. DiStefano and E. M. Johnson, PrQ~ ad. S~i lJSA, I~Li NGF receptor truncation also has been demonstrated in~ivo, where high levels of NGF-Rt were observed in the urine and plasma of neonatal rass followed by reducsd levels in the adult. P. S. DiStefano and E. M. Johnson, PrQC. Natl.A~ad. Scill~, l~
Furthermore, after sGiatic nerve transection in the adult (when Schwann cell N~iF
10receptors are induced) NGF-Rt levels are significantly elevated for prolonged psriods in the urine. Coupled with in vitrQ 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.
. ~' It would ba advantageous to provide an assay to determine the regulation and characterization of NGF-Rt in human urine during the course of development. Suchan assay could serve to detect NGF-Rt marker as an indication of abnormal development, regenera~ion and degeneration of peripheral nerves and quantitate 2 0 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 ot 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-2 5 site assay heretofore not possible with previously existing reagents.

$ummary of the Invention The present invention provides monoclonal antibodies or fragments thereof which specifically bind to human nerve growth factor receptor and human truncated 3 0 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 3 5 monoclonal antibody VIID1), VIIIC8 (A.T.C.C. deposit No. ~RL10619, producing : -monoclonal antibody VIIIC8) and XIF1 (A.T.C.C. deposit No. CRL10620, producing monoclonal antibody XIF1 j.

SUBSTITUTE ~HEET

~VO 92/n9631 PCI/US91/08968 Thc present invention also provides an assay to determine ~he 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 fac~or receptor, and which do not significantlybind to rat or chick nerve growth factor receptor has been bound, to form a rnixture.
This mixture is incubated for a time and under conditions sufficient for l O antigenlantibody complexes to form. The so-formed complexes then are contacted with an indicator reagent comprising a monoclonal antibody or fragment thereof which specincally 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 recep~or 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 bydetecting the measurable signal generated. The amount of human nerve growth factor 2 0 receptor present in the test sample, thus the amount of human narve growth factor receptor captured on the sol~d phase, 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 ~est samples.
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- E~ D~,~Pn of the Drawinp~
3 0 FIG. 1 is a photograph of an autoradiograph on SDS-polyacrylamide gel.
Standards are shown in lane 1 and include the following 14-C-methylated proteins:
myosin t200,000), phosphorylase B (92,500), bovine serum albumin (69,000), ovalbumin (46,000) and carbonic anhydrase (30,000). A sample of E9b conditioned medium (100111) was incubated with 4 nM 1251-NGF (lane 2) or 3 5 radiolabeled NGF in the presence of a 1 ~0-fold excess of unlabeled NGF (lane 3j.
. .
FIG. 2 is a photograph of an autoradiograph in which each of the monoclonal antibodies of the invention ~5 ~,lg) were incubated with NGF receptor affinity-cross-SUBST7TUTE SHEET :

WO 92/09631 PCI`/US91/08~68 ~

~inked 1O 1251-NRF ~100 ~LI) and solubilized from Esb cells (3 x 108). Standardsinclude the following 14C-me~hylated proteins: myosin (2oo~soo)~ 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 1251 affinity labeled NGF-Rt (2.2 j~l9 protein) purified approximately 650-fold from E9b condi~ioned mediurn by chromatography on ME20.4 sepharose and affinity labeied with l251~NGF.
1 0 Standards are those described in F~G. 2.

FIG. 4 is a photograph of an autoradiograph in which each o~ the monoclonal antibodies of the invention were incubated with 1251-NGF cross-linked NGF-R
contained in solubilized preparations from SH-SY5Y cells. The standards are those l 5 described in FIG. 2.
' FIGS. 5A-B are photographs of immuno~lots 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. 53 is a photograph of the immunoblot of samples mixed with 5% beta-mercap1Oethanol and boiled prior to resolution on gels and immunoblotting (reduced) .

FIGS. 6A-~D are bar graphs of compeUtion studies wherein the counts (cpm x 10~3/well) of each monoclonal antibody labeled with 1251 were plotted against NGF-Rt bound to immobilized unlabeled antibody, as indicated on the graphs.
FIG. 6A is a graph of 1251-lllG5.
3 0 FIG. 6B is a graph of 1251-VIID1.
FIG. 6C is a graph of 1251-VIIIC8.
FIG. 6D is a graph of 1251-XIF1.

FIGS. 7A-B are ~bar graphs of two-site RiSA using 1251-labeled IIIG5 3 5 mor,ioclonaJ antibody of the invention to detect NGF receptor and NGF-Rt bound ~o immobilized antibodies. Data are expressed as mean ~1- 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 (.. ).
SUBSTITUT~ SHEE~
. -:, ~WO 92/09631 PCI/US91/08968 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 inparallel 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 l251-lllG5 to increasing amounts of NGF-Rt immobilized on a solid support by antibody XIF1.
FIG. ~B 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 o~ 1251-lllG5 bound per well plotted versus ng of purified recombinant human truncated receptor protein (rhNGF-Rt).
FIG. 9B is a graph of the counts per minute of 1251-lllG5 bound per well versus urine volume added to the assay. Urine sample from a 6-year old male was 2 0 diluted with HEPES to a final volume of 50 111.
.:
FIG. 10 is a graph of urine NGF-Rt plotted as a Sunction of age in humans. The insert shows an expanded x-axis semi-log plot of urine NGF-Rt versus age. The value at t=0 was determined by extrapolation to the y-axis. Values were axpressed 2 5 as the fraction of t=0.
..
FIG. 11 is a graph of urine NGF-Rt versus age, expressed as per mg urine protein .

3 0 Detalled DescriptiDn-~-f--the Invention The present invention provides novel cell lines (hybr~domas) which produce tsecrete) monoclonal antibodies to NGF receptor, imrnunoassays which use the ; ~ monoclonal antib:dies, and kits which contain these monoclonal antibodies. These cell lines are Identified as cell line IIIG5 which produces monoclonal antibody 111G5, 3 5 cell line V11D1 whlch produces monoclonal antibody V11D1, cel1 line YIIIC8 which pr~duces monoclonal an~ibody VIIIC8, and oel! line XIF1, which produces monoclonal i antibody XIF1. These four ceil lines have been deposited at the American Type CulturQ~ Collection, 12301~ Parhlawn Drive, Rockville, Maryland, USA, as of ~ - SU~;STIT"T': SHFET
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WO 9~/09631 PCI'~US91108968 December 3, tg9û, under the following deposi~ numbars: cell line IIIG5 has been accorded A.T.C.C. Deposit No. CRL10617: cell line VIID1 has been accorded A.T.C.C.
Deposit No. CRL1û618; cell line VIIIC8 has been accorded A.T.C.C. Deposit No.
CRL10619; and cell line XIF1 has been accorded A.T.C.C. Deposit No. CRL10620.
The monoclonal anlibodies 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-10 receptor. We have discovered a distinct developmental regulation ot NGF receptor inhuman urine which is similar to that seen in the rat. P. S. DiStetano and E. M.
Johnson, PrDcl~atl. Acad. ~ci ~ISA, Ibid. No sexual dimorphism was evident at any time during development, nor was there any diurnal variation associated with NGFreceptor truncation in the adult. The presence of NGF receptor truncation correlates 15 well with the development of function in peripheral nerves, strengthening thehypothesis 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.
For example, in a first assay ~ormat, a capture reagent comprising a first monoclonal antibody or fragment thereof which speciflcally 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 2 5 both NGF receptor and truncated NGF receptor, to form a mixture. This mixture is incubated for a time and under conditions sufficient to form antigenlantibody complexes. These complexes then are contacted with an indicator r0agent which comprises a second monoclonal antibody or fragment thereof specific to a NGF
receptor and truncated nerve growth factor receptor to which a signal generating3 0 compound has been anached, to form a second mixtura. This second mixture is incubated for a ~ime and under conditions sufficient to form anubody/antigen/antibody complexes. The presence of NGF receptor and/or ~:
truncated nerve growtH ~factor receptor ~n ~the test sarnple which is captured on the solW phase, i f any, is determined~by ~de~eoting the measurable signal generated by the 3 5 signal generating compound. The amount of NGF receptor present is proportional to the signal generated.

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SUi3STllUTE;S~EEl ~

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~/0 9~/09631 P~/US91/08968 It is preferred that the monoc~onal antibody designated XIFl be used as the capture antibody, and that monoclonal antibody designated as lilGS, 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.
S
Alternatively, a monoclona~ 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 combinathn 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/antigerJantibody complexes. The presence, if any, of NGF receptor in the test sample and captured on the solid phase, is determined by detecting the 1 5 measurable signal generated by the signal generating cornpound. The amount of Nt3F
receptor present in the test sample is proportional to the signal genPrated.

In another assay format, one or a combination of more than one monoclonal antibody of the invention or frag~ent thereof, is employed as a comp0titive probe for 2 0 the detection of antibodies to NGF receptor and/or truncated NGF receptor. For example, NGF receptor and/or truncated NGF receptor cah be coated on a solid phase.
A test sample suspected of containing antibody to NGF receptor andlor truncated nerve growth factor receptor then is incubated with an indicator raagent comprising a signal generating compound attached to one or a combination of more than one 2 5 monoclonal antibody of the invention or fragment thereof, for a time and under condition 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 signal3 0 generated from a confirmed negative NGF receptor and truncated NGF receptor test sample indicates the presence of anti-NGF receptor antibodies and~or anti-tnmcated NGF receptor antibodies in the test sample.
.
Although solid-phase assays have been described, the assays described 3 5 hereinabove atso can be performed in solution, not employing~ sotid-phase ~ ~ -technologies.
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SUBSTITUTE SHEET

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WO 92/~9631 P~r/US91/08968 In yet ano~her detection method, each of the monoclonal antibodies of the present invention can be employed in the detection of NGF rec0ptor and/or truncated NGF receptor in fixed or fresh tissues or cells by immunochemical analysis.

In addition, 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 preser~t inven~lon can l O be provided individually to detect NGF receptor and/or truncated NGF receptor.
Combinatisns of the monoclonal antibodies (and fragments thereot) provided herein also may be used together as components in a mixture or "cocktail" of anti-NGF
receptor antibodies, with different binding specificities.

It also is contemplated that 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 ba used in the assay formats should specifically bind to NGF
receptor and/or truncated nerve growth factor receptor. The polyclonal antibody 2 0 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 cochtail of polyclonal antibodies.

2 5 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, salwa, 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 ~nclude the walls of wells 3 0 of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particlest Sepharose-like beads,and others. -~
The indicator reagenl comprises a signal generating compound (label) which 3 5 is capable of generating a measurable signal detectable by external means conjugated (attached) to a specific binding member for NGF receptor andtor truncated NGF
receptor. "Specific binding member" as used herein means a member of a specific binding pair. Thàt is, ~wo di~feren~ molecuies where one of the molecules through ~ .
SUBSTI~UTE SHEET

~0 92/()9631 PCI-/US91/089~8 chemical or physical means specifically binds to the second mo~cule. In addi~ion to being an antibody member of a specific binding pair for NGF receptor and/or truncated nerve grc~wth factc>r receptor, the indicator reagent a~so can be a member o~ any specific binding pair, including either hapten-anti-hapten systems such as S biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a re~eptor molecule, an enzyme cofactor and an enzyme, an enzyme inhibitor or an enzyme, and th~ 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
10 receptor as in a sandwich assay, to the capture reagent as in a cornpetitive 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 fluorescein15 and rhodamine, chemiluminescent compounds, radioactive elements, and direct visual labels. Examples of enzymes include alkaline phosphatàse, horseradish peroxidase, beta-galactosidase, and the like. Examples of radioactive elaments include 1 251, 3H and 35S. 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 2 O more additional substances.
:
It is contemplated that the reagent(s) employed for the assay, such as the monoclonal antibodies of the invention, and various washing and assay reagents, can be provided in the form of a kit with ~one or more containers such as vials or bottles 2 S containing a separate reagent such as a monoclonal antlbody The following examples demonstrate the advantages and utility of this invention for serodiagnosis of NGF receptor and truncated NGF receptor by describing methods for the development, characterization, and clinical utllity of the 3 O mo~oclonal antibodies and assay of the invention. These examples are meant to illustrate, but not to limit, the spirit and scope of the invantion.

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' SUBSTITL~E SHEET

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WO 9~/0~631 PCr/lJS91/Og968 ~

~AMPLES

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 ~L~, ~ien~Q 232:518-521 11986]). Conditioned medium was decanted from the cells, brought to 0.02% with sodium azide and stored at 4C. An immunoaffinity chromatography resin was prepared by coupling affinity purified monoclonal antibody ~/iE20.4 (A. H. Ross et al., Proc. NalL~, $~ ~ 81:6681-6685 (198~) to cyanogen bromide activate Sepharose-4B (4 mg antibody/ml resin, available from Pharmacia, LOCATION), according to the method of R. Axen ~Lal"
214:1302-1304 (1967). 500 ml of E9b conditioned medium was passed over the ME20.4-Sepharose column. The column was washed with PBS (20 mM
phosphate buffered saline, pH 7.4) and eluted with 4 M MgCI2 in acetate buffer (0.2M, pH 6.5). This resul~ed in an approximate 100-fold purification of NGF-Rt from E9b conditioned medium. The column eluate was extensively dialyzed against - PBS and concentrated prior to use in immunization. A two-month old BALB/cByJ
mouse received a primary intraperitoneal (IP) injection of partially purified Rt(approximately 100 ~9 total pro~ein) in MPL+TDM adjuvant (avallable from RIBI
Immunochem. Res. Inc., Hamilton, MT), followed by a booster injection containingthe same amount of protein at three (3) weeks. The mouse was bled by retroorbital puncture prior to the primary immunization and at ten (10) days following the boost. An intravenous (IV) injection of approximately 40 ~Lg total protein was 2 5 administered seven (7) weeks after the three (3) week boost. Three (3) days later, ~ `
spleen cells were fused with NS-1 mouse myeloma cells according to establi~hed procedures (see G. Kohler anci C. Milstein, Dl~. 256:495-497 11975]; a. Kohler and C. Milstein, Eur. J. Immunol. 6:511-519 [1976]; and V. T. Oi and L. A.
Her~enberg, in B. B. Mishell and S. M. Shiigi, eds., ~ua~
3 0 Imrmmolo~y, W. H. Freeman, San Francisco, pp. 351-371 [19801). Following selection with hypoxanthine, aminopterin and thymidine (HAT), th0 hybridomas were maintain~d 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 IlM) and n-(2-3 5 hydroxyethyl)1piperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.3 (10 mM). .

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SUBSrl~UTE SHEET

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- . : ' : ' ~ ., .. ' ! . . , ~WO 92/09631 PCI/US91/08968 Example 2 Affinity Labeling of NGF-R With 1251-NGF
Mouse submaxillary NGF was purified according to the procedure of V.
Bocchini and P. U. Angelleti, Pro~,NatL,~cadA Sci. USA 64:787-794 (1969), and was labeled with 1251 according to the me~hod of J. J. Marchalonis, Bi~h~
113:177-190 (1969). Affinity labeling of cell surface and truncated NGF-R
species was performed as described previously by J. Taniuchi et at., e~
Sçi. !1~9 83:4094 4098 (1986); P. S. i~iStefano and E. M. Johnson, l~lcL~L
8:231-241 (1988); and P. S. DiStefano and ~. M; Johnson, Proc. Natl~ A~L Sci.
1 0 USA 85:270-274 (1988).

Example 3 Radiometric Immunosorbent Assay (RISA) Hybridoma supernatants were screened for the presence of antibodies using 1 5 the RISA tesl described by E. A. Pierce et al., ~o~b~ 153:67-84 (1986).
Briefly, Immulon 2 Removawell strips (available from Dynatech Labs, Alexandria, VA) were coated with goat anti-mouse IgG (50 jli, 50 ~Lg/ml) in PBS (pH 8.0) overnight at 4C or at room temperature for one (1) hour. After removai of the goat ànti-mouse IgG, the wells were blocked with 1.5% BSA (150 tlg/ml) in PBS for 30 2 0 minutes at room temperature. The wells were washed three (3) times with cold PBS
followed by the addition of hybridoma supernatants (50 jll) 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 ~o 1251-NGF
(approximately 25,000 cpm) was added to each well and incubated for 45 minutes at 2 5 4C. The wells were washed four (4) times with cold PBS containing 0.05% Tween-20~9 (available from Sigma Chem. Co., St. Louis, MO), and the wells were analyzed for the presence of radioactivity.
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Example 4 ;
3 0 Immunoprecipitation of NGF-R and NGF-Rt Receptor-containing samples affinity labeled wi~h 1251-NGF (50 to 100 ~
were incubated with~hybridoma supernatant (50 to 100 ,ul), mouse serum (50 jul of a 1 :100 dilution in PBS) or purified monoclonal antibodies (5 llg) for two to four hours at 4C. A suspension of goat anti-mouse IgG Sepharose in PBS was added 3 5 (10%, vlv), and the mixture was incubated for one hour. Sepharose in ~ubes con~aining NGF-R was washed twice with 500 jul of PBS containing 0.05% Tween-20, whereas Sepharose in tubes containing NGF-Rt was washed with PBS containing 0.5/O bovine serum albumin (BSA), 0.~% sucrose and Q.1% Tween-20~. The SUBSr~ E ~i;HEE~
.

WO 92/09S31 P~r/us91~0~968 washed resin was either counled for radioactivity or mixed with reducing SDS-sample buffer. Sample buffer was incuba~ed with resin for one hour at room temperature and was removed by centrifugation. Samples were boiled for 90 seconds and applied to 10% discontinuous SDS-polyacrylamide gels. Electrophoresis was S performed using the buffer system of P. H. O'Farrell, ,l. Biol. ~hem. 250:4007-4021 (1975). Dried gels were exposed to Kodak XAR film using an intensifying screen at -70C. Analysis of NGF R or NGF-Rt after affinity cross~linking to 1251-NGF, immunoprecipitation and gel electrophoresis is hereinafter referred toas cross-link immunoprecipitation, or Cl IP assay.

Example 5 Radiolabeling of Monoclonal Antibodies Affinity purified antibodies (50 llg) were radiolabeled with 1251-Bolton-Hunter reagent (2200 Ci/mmole, 1 mCi) according to the procedure of Bolton and Hunter (A. E. Bolton and W. M. Hunter, ~ ,L 133:529-539 [1973]). 1251-labeled antibody was separated from unreacted 1251-Bolton-Hunter reagent by chromatography on an Econopac 1-DG column (Biorad Labs, Richmond, CA), equilibrated in PBS containing 0.2% gelatin and 0.01% NaN3.

2 0 Example 6 Immunoblotting NGF-R was solubilized from E9b cells using 20 mM sodium phosphate buffer (pH 7.4) containing 2% n-octyl glucoside, 0.65 M NaCI, 1 mM PMSF and 1 mM -iodoacetamide. The solubilked preparation was mixed with either SDS-sample 2 5 buffer containing 5% beta-mercaptoethanol followed by boiling for 90 seconds, or with non-reducing SDS-sample buffer and used directly without boiling. Samples were resolved on 10% discontinuous SDS-polyacrylamide gels. Prestained, reduced molecular weight markers (available from BioRad Labs, Richmond, CA) were run in a parallel lane. Protein was transferred electrophoretically to nitrocellulose 3 0 membranes as described by H. Towbin et al.. Proc. Na~l"~cad. Sci. USA 76:4350-4354 (1979). Membranes were incubaled for one hour at room ternperature in PBS containing ~% 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 ,ugiml) in the same buffer. Membranes were rinsed three 3 5 times each for five minutes wi~h PBS containing 0.5% non-fat dry milk and 0.05%
Tween-20. Membranes then were incubated for two hours in the presence of 1251-- labeled goat-anti-mouse IgG. Radiolabeled antibody was diluted in PBS ( 3 x 106 cpmlml) containing 0.5% non-fat dry milk and 0.05% Tween-20, and one with PBS
SUBSTlfUTE SHEE~

~WO 92/09631 P~ S91/~18968 ~ 3 alone. Dried membranes were exposed to Kodak XAR nlm using an intensifying screen at -70C until films were developed.

Example 7 Antibody Competition Studies Affi`nity purified antii~odies were radiolabeled with 12~1-Bolton-Hunter reagent. Cells (E9b or Ltk-) were so~ubilized as described hereinabove. N(~R-Rt in E9b cell conditioned meciium was used directly or was purified by immunoaffinitychromalography on a resin conslructed 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.~5 M NaCI, PBS, 50 mM CAPS buffer ~pH g.8). NGF-Rt was eluted from the column using CPAS buffer (pH 11.5). Column fractions werebrought to pH 7.4 by the addition of 1 M HEPES buffer, pH 7Ø

Immulon 2 Removawells were coated with ~o ~l of a given monoclonal antibody of the invention (~0 Ilg/ml) in PBS overnight at 4C. Additional binding sites were blocked with the addition of 150 1ll of 1.5% BSA in PBS for 30 minutes at room temperature. The wells were washed three times with cold PBS, after which 50 ~11 of the receptor-containing preparation was added and incubated for 90 2 0 minutes at room temperature. The weils were washed three times with cold PBS
followed by the addition of 50 11l of a given 125i-labeled monoclonal antibody (2.5-5 x 106 cpm/well) and incubation for 45 minutes at 4C. The wells then were washed four times with PBS containing 0.05JO Tween-20 and counted for radioactivity .
Example 8 General Procedures Protein concentration was determined by the method of M. M. Bradford, Anal.
h~ 72:248-3454 (1976) using crystalline BSA as a standard. Laser 3 0 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).
Antibodiès were~isotyped using a ScreenTypeTM kit obtained from Boehringer Mannhein ~Indianapolis, IN).
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WO 92~09631 ` P~US91~û8968 Example 9 Monoclonal Antibody Production Monoclonal antibodies were generated against human recombinant NGF-Rt as follows. Briefly, the Esb cell line,~described by M. V. Chao ~" Science 232:518-521 11986]), a mouse L cell transformed with hurnan genomic DNA
which expressed the NGF recep~or gene, was maintained in culture and NGF-Rt was harvested from 3-4 day conditioned medium. The NGF-Rt from the conditioned medium of E9b calls was used as a source of receptor for immunization. To demonstrate that NGF-Rt from E9b conditioned medium was similar to that reportedpreviously from other sources, receptor in E9b conditioned medium was affinity labeled with 1251-NGF, immunoprecipitated with antibody ME20.4 and resolved on SDS-polyacrylamide gels. This resulted in the appearance of a labeled protein with the expected apparent molecular weight of 66,000 daltons, as shown in FIG. 1. NGF- -Rt was immunopurified from E9b conditioned medium and used to immuni e a BAiB/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.

2 0 Hybridomas which secreted an~ibody 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 imrnunoprecipitation, and 18 of 34 wells remained positive for ~he presence of antibody to the NGF-R using this assay. Five cell lines continued to screen positive 2 5 for antibody to NGF-R ah~er expansion of the cell lines, and these were cloned by limiting dilution. After clon~ng and expansion, four hybridoma lines remained positive for antibody to NGF-R. These hybridomas are identified as cell line IIIG5 which produces monoclonal antibody lliG~, cell line VIID1 which produces monoclonal antibody VIID1, cell line VIIIC8 which produces monoclonal antibody 3 0 VIIIC8, and cell line XIF1, which produces monoclonal antibody XIF1.
Characterization of these monoclonal antibodies using class- and subclass specific anti-mouse immunoglobulin antisera in an ELISA showed that all were IgG1,k antibodies. ~ : -,.
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~0 92/09631 PCI`/US91/08968 Example 10 Anaiysis of Antibodies Using CLIP
Monoclonal antibodies were tested for the ability to immunoprecipitate 1251-NGF-affinity labeled receptor. Samples were resolved on SDS-5 polyacrylamide gels to confirm the molecular identity of the immunoprecipitated species. Immunoprecipitation of affinity labeled receptor from E9b cells by all four monoclonal antibodies of the invention (designated as IIIG5, VIID1, VIIIC8, and XIF1) resulted in the appearance of a 90,000 Mr species on gel autoradiograms, as shown in FIG. 2. Subtracting a monomer of N(3F (13,000 daltons) results in a net molecular weight of approximately 80,000 daltons for the cell sur~ace receptor.
The same protein was immunoprecipitated using the monoclonal antibody ME20.4 to human melanoma cells NGF-R (A. H. Ross et al., PrQc. Natl, ~.a~. ~L~.~ 81:66~1-6685 ~1984~. No affinity labeled receptor was immunoprecipitated when either a control murine IgG1,k antibody (MOPC21) or a monoclonal antibody specific for the rat NGF-R (Ab 192) was used (according to C. E. Chandler et al., J. ~QI. Chem, 259:6882-6883 [19841). The four monoclonal antibodies produced were capabie 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 1251-NGF-NGF-Rt complex was not immunoprecipitated by the antibody MOPC21.

In order to determine whether the antibodies recognized both !he high and low affinity form of the NGF-R, the CLIP assay as described in Example 4 was performed 2 5 using affinity labeied 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. L
Nellrosci. 5:1717-1728 E1985~). All four monoclonal antibodies of the invention,as well as the antibody ME20.4, immunoprecipitated the 90,000 Mr NGF-R1251-NGF-R complex from SH-SY5Y cells receptor, as shown in FIG. 4. NQ labeled 3 0 material was immunoprecipitated by MOPC21.

Example 11 ; Species Cross-Reactivity Using the~two-site RiSA~as described in Example 3, all of the monoclonal 3 5 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.
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WO g2/09631 PCr/us~l/08968 4 Example 12 -Immunoblot of the Cell Surface NGF-R
In order to further characterize the specificity of antibody binding, immunoblotting studies were performed using NGF-R solubilized from Esb cells.
When receptor samples were prepared in the absence of reducing agent and were not boiled prior to resollJtion on gels, two of the monoclonal antibodies (VIID1 and XIF1) bound to a major protein species with a molecular weight of approximately 68,000daltons, as shown in FIG. 5A. No immunoblotting occurred with the control antibody MOPC21. Additional experirnents indicated that if long enough film exposures were used, all of the antibodies of the invention immunoblotted NGF-R; however, the intensity of the autoradiographic signal obtained was always less for monoclonalantibodies IIIG5 and VIIIC8. When receptor samples were exposed to a reducing agent ~beta-mercaptoethanol) and boiled prior to electrophoresis, there was no antibody binding, as shown in FIG. 5B.
Example 13 Epitope Mapping and Selection of Monoctonal Antibodiss for Assay Solid phase competition studies were performed to determine whether the monoclonal antibodies of the invention bound to distinct receptor epitopQs. All of the 2 0 monoclonal antibodies were tested on the solid phase, as well as serving as the radiolabeled ~top~ monoclonal antibody. In this assay, the retention of radiolabsl 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. The results of the competition studies clear!y 2 5 showed that antibody IIIG5 bound to an epitope of the NGF-Rt that was distinct from ::
the epitopes recognized by monoclonal antibodies VIID1, VIIIC8, XIF1 and the previously developed monoclonal antibody ME20.4, as shown in FIGS. 6A-D. When each monoclonal antibody was probed against itself. the retention of radioactivity in the wells was no greater Ihan when a control antibody MOPC21 was used. These 3 0 results indicated that the monoclonal antibodies of the invention did not recognize repeated epltopes on the receptor.

Two monoclonal antibodies recognizing distinct sites on the receptor were selected for use in the development of a two-site radiometric immunosorbent assay.
3 5 Based on ~results df the previous competition studies, antibodies l~liG5 and XIF1 were chosen. When either NGF-R ~as shown in FIG. 7A) or NGF-Rt (as shown in FIG. 7B) was assayed, the combination of radiolabeled IllG5 with XIF1 as the anchoring (capture) antibody resulted io the maximum retention of specific counts per well.
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~0 g2/09631 PCI/US91/089~8 ~ 7 Ltk cells or Ltk- cell conditioned medium were assayed in parallel with E9b cells and E9b cell conditioned medium to assess non-specific binding. These studies showed that non-specific binding in the assay was higher when solubilized cell preparations were used, as compared to conditioned medium. The use of MOPC21 as .
S the anchoring (capture) antibody also provided a good assessment of non-specific binding.

In order to test the linearity of ~he assay, serial dilutions of NGF-Rt that hadbeen concentrated by ammonium sulfate precipitation were tested. There was a 10 linear relationship between the amount of NGF-Rt added and the amount of 125l-IIIG5 retained per welt, up ~o at least 30,000 cpm per well, as shown in FIG. 8A.
Data obtained from the two-site RISA according to Example 3 were compared to those obtained using the CLIP assay of Example 4. FIG. 8 shows a ciose correlation (R-0.998) between the relative values obtained using the two-site RISA and the CLIPassay. ' ~ "

E~ample 14 Sample Collection and Preparation Urine samples were collected from 70 normal human subjects ranging in age 2 0 from 1 month to 68 years. Urine from 4 pregnant women in their third trimester (age range 33-41 years) also was collected. Urine was collected in polypropylenespecimen containers (available from Scientific Products, McGaw Park, IL), immediately placed on ice, and frozen at -80C wilhin 2 hours of collection. Urine samples were routinely assayed within 2 weeks of collection. There was no reduction 2 5 in assay values for NGF-Rt or for creatinine when samples were frozen at -80C for at least three weeks whPn compared to fresh urine. For neonates, urine was collected from cloth diapers or by the use of U-bags (available from Hollister, Kirksville, MO). For assay, samples were thawed at 4C, centrifuged at 13,000 x 9 for 5 minutes, and diluted 1:1 with 0.5M HEPES buffer, pH 7.0, before addition to the 3 0 assay plates as described hereinbelow. Assay blanks used as negative controls ' .
consisted of 20 mM phosphate, 160 mM NaCI (phosphate buffered saline lPBS}) diluted 1:1 with HEPES buffer.

Example 15 .-: .
3 5 Creatinine and Protein .' ' The thawed and centrifuged urine samples were assayed for creatinine usin~ -the picric acid method according to N. W. Tietz, T~tbook of ~linica~b~mj~, W. B.Saunders Co., Philadelphia, pgs. 1278-1280 (1986). Protein was'determined on SUBSTlTlJlE SHEET - ~

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urine samples by the method of M. M. Bradford, ~aL~ m, 72:248-2~4 (1976), using bovine serum albumin as the standard.

Example 16 Two-Site RISA
A two-site radiometric immunosorbent assay (RISA) ~or nerve growth factor receptor (NGF-R) and truncated nerve growth factor receptor (NGF-R1) was developed using the monoclonal antibodies of the invention that bound to distinct 10 epitopes on the receptor protein. XIF~ was chosen as the anchoring ~capture) antibody and was used to bind NGF-R from solubili~ed cell preparations or NGF-Rt to the solid phase. 1251-lllG5 (5 x 1o6 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 ~L~. Anal. Bio~em. 153:67-74 (1986). Monoclonal antibody XIF1 was diluted to 50 ~Lg/ml in PBS (pH 8.0), and then added to Immulon Il Removawell strips (available ~rom Dynatech, Chantilly, VA) in a voiume of 50 and incubated overnight at 4C. Non-reacted sites on the wells were blocksd with2 0 1.5% bovine serum albumin for one (~) hour, and washed with PBS (pH 8.0) using a Dynatech plate washer (Dynawasher lIrM, available from Dynatech, Chantilly, VA).
Fifty (50) ,ul of diluted urine samples described in Example 14 were added to the wells in quadruplicate for one (1) hour followed by washing. Monoclonal antibodyIIIG5 was iQdinated by the lactoperoxidase method (J. J. Marchalonis, Biochem. J.
113:299-305 11969]) to specific activities of 4000-5000 cpm/fmole. 125l-IIIG5 (500,000 cpm) was added to each well for about 45 minutes on ice, and thenwashed four (4) times with ice-cold PBS containing 0.1% Tween-2û~9 (available from Sigma Chem. Co., St. Louis, MO). Wells were broken apart, placed in tubes and counted on a Beckman gamma counter (Model 5500) for one (1) minute. Counts per :
3 0 minute were always normalized to a standard sample of purified recombinant human - truncated receptor protein. Data were expressed as nanograms of NGF-Rt per ~Lg .
creatinine or as nanograms per mg of urine protein. In some experiments, saturable 51-lllG5 binding was performed on urine NGF-Rt anchored to XIF1 to assess IIIG5 i affinity to urine NGF-Rt. For these studies, IIIG5 was iodinated to specific activities - 3 5 of 600-800 cpm/fmol and binding was performed at various concentrations of labeled IIIG5 for 45 minutes as described hereinabove. KD and Bmax values were determined by Scratchard plot. Bmax was expressed as nanograms (ng) 1251-NGF
bound per llg Of creatinine which had been determined as described in Example 15.
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~ ~o 92/09631 PCr/US91/0896~

Example 17 Affinity Label/Cross-Link Immunoprecipitation (CLIP) of NGF-Rt NGF-Rt in urine samples (see Example ~4) of various aged sub~ects was affinity labeled with 1251-NGF (specific activity = 2000-2500 cpm/fmole) as - described previously by P. S. DiStefano and E. M. Johnson, Proc. Natl. Acad. Sci. USA, lki~. Immunoprecipitation of NGF-NGF-Rt complexes was achieved using either monoclonal antibody ME20.4 or XIF1. Samples were processed for SDS-PAGE and autoradio~raphy using Kodak X-0-MATTM film ~Kodak, Rochester, NY) as described 1 0 by P. S. DiS~efano and E. M. Johnson, Proc. Natl. A~d. Sci. USA, lki~. Bands appearing on gel/autoradiograms were quantitated with an LKB Ultroscan XL laser densitometer (available from LKB, Piscataway, NJ).

Example 18 1 5 Statistics Statistical differences in urine NGF-Rt values obtained by following the procedures described in Examples 16 and 17 from various groups, were datermined using one-way ANOVA followed by Newman-Keuls post-hoc analysis. Significant differences were determined at the .01 Ievel. Binding constants for ~251-NGF
2 0 binding to NGF-Rt were compared using the Students t-test.

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. As shown in FlG. 9A, the assay was linear with respect to the amount of purified 2 5 recombinant truncated receptor up to 50,000 cprn. The internal standard of E9b conditioned medium routinely registered 10,000 cpm. As shown in FIG. 9B, the assay also was linear with the amount of urine added (diluted 1:1 with HEPES
buffer) up to a volume of 25 ~LI. The sample-to-blank ratios ranged from 3:1 to 30:1, depending on the content of NGF-Rt in ~hè sample~ Analytical precision was 2-3 0 8% and inter-assay variability was less than 4%, as determined on the urine from .
17 subjects. In addition, NGF-Rt in adul~ 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 1wo months at -80C without experiencing loss of activity. It further was determined ~hat urine samples could be 3 5 left at room temperature for up ~o eight hours without loss of NGF-Rt activity or creatinine level. Boiling of urine samples, however, abrogated activity without affecting creatinine levels. The current throughput of the assay described in SUBSTITU~E 5~EEl . . -:
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WO 92/~9631 PCI`/US91/08968 Example 16 is such thal 65 samples can be assayed in quadruplicate by an individual in one day.

When absolute concentrations of urine NGF-Rt obtained by Example 16 were plotted as a function of age, there was a minor trend showing decreased levels with age. The absolute amounts of urine NFG-Rt ranged from 56-1200 nglml urine. As shown in FIG. 10, when expressed as ng NGF-RV~lg creatinine, there was a distinct and dramatic regulation of urine NGF-Rt as a function of age. At one month of age, the earliest time point examined, levels of NGF-Rt were very high, declining rapidlythrough the first year of life. Between 1-15 years of age, levels of NGF-Rt declined more gradually, reaching adult levels at 15 years of age. Between the age ot 15-68 years, values were essentially constant at 5% of the mean value obtained for onemonth subjects. Of the 70 normal subjects assayed, values for only ane individual fell outside the curve generated and shown in FIG. 10. This individual was re-1~ assayed six months afterwards, and found to lie in the normal range. Thus, the reliability of the assay is greater than 98%. The insert of FIG. 10 shows an expanded x-axis plot of the data in order ~o gain an estimation of the decrease in urine NGF-Rt during very early developrnent. During the first one to six months of life, urine NGF-Rt declined rapidly in urine. Between 0.5 to 3.5 years of 2 0 development, 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 .
2 5: 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 cievelopmental pattern was evident for NGF-Rt in human urine.

To statistically evaluate changes in urine NGF Rt as a function cf age and sex, the data from F!G. 10 were placed in age bins and the mean values of these groups were compared. In addition, the NGF-Rt levels observed in pregnant women were compared to normal aduits. These data are shown in Table 1.
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o 92/09631 3~ PCI`/US91/08968 ~ ~ . . ..

Analysis of Urine NGF-Rt Values S Age (Years~ NGF-~t ln~ cre~injne~ c 0.08-0.10 7.00 + 2.36~- . 5 3-3.5 0.91 + 0.23~ 5 15-68 (M ~ F pooled) 0.31 + 0.06 31 3 0 - 5 0 M 0.29 + 0.06 14 101 5-68F 0.34 + 0.06 17 33-41 (pregnant) 0.58 + o.09~ 4 .

~ Values from various age ranges were pooled and expressed as mean + S.D. with the number of observations in each 3roup indicated on the right. M, male; F, female.Urines were sampled from women at 7.5-8.5 months of pregnancy.
p~.01 compared to all other groups.

Monoclonal antibodies have been produced against a soluble, truncated form of 2 0 the human NGF-R elaborated by Esb 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. ~ç~SA 85:270-274 [1988]) and melanoma cells (A. A. Zupan et al., J. Biol. ~h~ 264:11714-l1720 [19891) with respect to molecular weight and displacement of 1251-NGF binding by unlabeled NGF. Although2 5 t he four monoclonal antibodies of the invention immunoprecipitate affinity labeled receptor species to varying~ degrees, it is evident that they all lecognize the whole !cell surface) and truncated forms of the NGF-R. Additionaily, all antibodies bind to the high affinity form of the~ NGF-R localized on the cell surface ~SH-SY5Y human neuroblastoma cells. This cell line has been reported to express exclusively the high 3 0 affinity form of the NGF-R (K. H. Sonnenfeld and D. N. Ishii. .I. N~Ur~ci~ 5:1717- -1728 (1985). The fact that the monoclonal antibodies of the invention were generated agalnst truncated, low affinity type receptors and also recognize highaffinity receptors suggests !hat low and~ high affinity NGF-R's have overlappingregions of sequence homoiogy or represent the same protein. Evidence suggests that 3 5 high affinity is conferred on the core (low~ affinityj Nt;F-R protein by association with a modulatory protein localed within ihe.plasma membrane (see, for example, M. Hosang and E. M. Shooter, J. Biol. Chem. 260:655-662 [198~5~; S. H. Green and - -SUBSTlTU'rE 8~

:

WO 92/~9631 Pcr/vssl/o8s68 L. A. Greene, J. Biol. Chem. 261:15316-15326 [1986]; and B. L. Hempsted et al., Science 243:373-375 l1 989]).

The monoclonal antibodies of the invention produced in this study were screened on 1he basis of their ability to bind human NGF-R affinity labeled with1251-NGF in both a solid phase and an immunoprecipitation assay. The monoclonal antibodies of the invention all cross-react with truncated NGF receptor from monkey but not rat or chick. Thus, the monoclonal antibodies of the invention specifically bind to human nerve growth factor receptor and human trunca~ed nerve growth 1 0 factor receptor, and also specifically bind to monkey truncated nerve growth factor receptor, and do no~ significantly bind to rat or chick nerve growth factor receptor.
Two other reports have described the production of monoclonal antibodies to the human NGF-R, none of which cross react with receptor from chick or rat (see A. H.
Ross ~aL, ~ 81:6681-6685 11984] and N. Marano ~Lal., 1 5 ~, Neluo~hen~, 48:2~5-232 [1987]). A comparison of NGF-R cDNA from human, rat and chicken shows that the encoded receptor amino acid sequence is highly conserved (see, for example, D. Johnson et a~ I 47:545-554 [19861; M. J.
Radeke ~LaL, Nature 325:593-597 [1~871; P. Enfors et a!., NeurQrl 1:983-996 ~1988l; E.Escandon and M. V. Chao, Dev. Brain Res. 47:187-196 l1989l; and T. H.
Large et al., Neur~ 2:1123-1134 [1989]). This may explain why the generation of antibodies that cross-react with a variety of species has been difficult.
, ~'.
The immunoblotting experiments described herein demonstrated that the -monoclonal antibodies of the invention did not bind to reduced forms of the NGF-R
2 5 after transfer to nitrocellulose. This suggests that the antibodies recognke NGF-R
species in a conformation dependent fashion. We have determined through additional experiments that the antibodies bind to receptor expressed in a bacterial system, implying that th~ey recognize !he core protein rather than sugar moieties present on the NGF-R as those previously described by P. M~ Grob Qt al., J. Biol. Chem~
3 0 260:8044-8049 (1 985).
: .
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 3 5 i n 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 advan~age over previously described methods, because NGF receptor can be assayed in ~he absence of ligand (NGF). Measurement of SUE~STITUTE S~E~T

WO ~2/09631 ~?~, PCr/US91/08968 NGF^Rt using the two-site RISA and the CLIP assay yielded similar results. Ths two site RISA also was used to examine the regulated excretion of NGF-Rt in human urine during development. Antibodies that bind to different receptor epitopes provide important tools which can be used to confirm the specificity of antibodyS binding to receptor in tissue sections using immunohistochemical techniques. In conjunction with the over-expression of recombinant receptor, these novel monoclonal antibodies also can be used to develop immunoaffinity purification strategies to facilitate studies of NGF-R structure.

OSher modifications and variations of the specific embodiments of the invention as set forth herein will be apparent to those skilled in the art.
Accordingly, the invention is intended to be limited in accordance with the appended claims.

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Claims

WE CLAIM:

1. 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.

2. The monoclonal antibody of claim 1 wherein said monoclonal antibody has the binding specificity of the monoclonal antibody derived from a hybridoma selected from the group consisting of hybridoma A.T.C.C. deposit No. CRL10617, hybridoma A.T.C.C. deposit No. CRL10618, hybridoma A.T.C.C. deposit No. CRL10619and hybridoma A.T.C.C. deposit No. CRL10620.

3. A hybridoma which produces a monoclonal antibody or fragment thereof which specifically binds to human nerve growth factor receptor and humantruncated nerve growth factor receptor, and which also binds to monkey truncatednerve growth factor receptor, and which does not significantly bind to r t or chick nerve growth factor receptor.

4. The hybridoma of claim 3 having the identifying characteristics of a hybridoma selected from the group consisting of hybridoma A.T.C.C. deposit No.
CRL10617, hybridoma A.T.C.C. deposit No. CRL10618, hybridoma A.T.C.C. deposit No. CRL10619 and hybridoma A.T.C.C. deposit No. CRL10620.

5. A monoclonal antibody identified as IIIG5 produced by the hybridoma on deposit with the A.T.C.C. and assigned A.T.C.C. deposit No. CRL10617.

6. A monoclonal antibody identified as VIID1 produced by the hybridoma on deposit with the A.T.C.C. and assigned A.T.C.C. deposit No. CRL10618.

7. A monoclonal antibody identified as VIIIC8 produced by the hybridoma on deposit with the A.T.C.C. and assigned A.T.C.C. deposit No. CRL10619.

8. A monoclonal antibody identified as XIF1 produced by the hybridoma on deposit with the A.T.C.C. and assigned A.T.C.C. deposit No. CRL10620.

9. A method for determining the presence of human nerve growth factor receptor and/or human truncated nerve growth factor receptor in a test sample which may contain human nerve growth factor receptor or human truncated nerve growth factor receptor, comprising:

a. contacting the test sample with a monoclonal antibody or fragment thereof attached to a solid phase which antibody 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, to form a mixture;

b. incubating said mixture for a time and under conditions sufficient to form antigen/antibody complexes;

c. contacting said complexes with an indicator reagent comprising a signal generating compound capable of generating a measurable detectable signal attached to a second 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, to form a second mixture;

d. incubating said second mixture for a time and under conditions sufficient to form antibody/antigen/antibody complexes;

e. determining the presence of human nerve growth factor receptor or human truncated nerve growth factor receptor in the test sample by detecting themeasurable signal generated.

10. The method of claim 9 wherein said signal generating compound is selected from the group consisting of a luminescent compound, a chemiluminescentcompound, an enzyme and a radioactive element.

11. The method of claim 9 wherein said monoclonal antibody or fragment thereof of steps (a) or (c) specifically binds to an epitope on a molecule of approximately 50,000 daltons or approximately 80,000 daltons.

12. The method of claim 9 wherein said monoclonal antibody or fragment thereof attached to the solid phase of step (a) or said indicator reagent of step (c) has the binding specificity of the monoclonal antibody produced from the hybridoma selected from the group consisting of hybridoma A.T.C.C. deposit No. CRL10617, hybridoma A.T.C.C. deposit No. CRL10618, hybridoma A.T.C.C. deposit No. CRL10619and hybridoma A.T.C.C. deposit No. CRL10620.

13. The monoclonal antibody of claim 9 wherein said monoclonal antibody or fragment thereof of step (a) is identified as the XIF1 monoclonal antibody produced from the hybridoma cell line on deposit with the A.T.C.C. and assigned A.T.C.C. deposit No. CRL10620.

14. The monoclonal antibody of claim 9 wherein said monoclonal antibody or fragment thereof of step (c) is identified as the IIIG5 monoclonal antibody produced from the hybridoma cell line on deposit with the A.T.C.C. and assigned A.T.C.C. deposit No. CRL10617.

15. An assay kit for determining the presence and/or amount of human nerve growth factor receptor or human truncated nerve growth factor receptor in a test sample, comprising:

a. a capture reagent comprising a monoclonal antibody or fragment thereof which specifically binds to human nerve growth factor receptor and humantruncated nerve growth factor receptor, and which also binds to monkey truncatednerve growth factor receptor, and which does not significantly bind to rat or chick nerve growth factor receptor;

b. an indicator reagent comprising a signal generating compound attached to 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.

16. The assay kit of claim 15 wherein said monoclonal antibody of (a) and (b) has the binding specificity of the monoclonal antibody produced from a hybridoma selected from the group consisting of hybridoma A.T.C.C. deposit No.
CRL10617, hybridoma A.T.C.C. deposit No. CRL10618, hybridoma A.T.C.C. deposit No. CRL10619 and hybridoma A.T.C.C. deposit No. CRL10620.