Specific antibodies against an epitope existing within the A/B region of estrogen receptor proteins

A unique monoclonal antibody is provided for determining the functional status of human estrogen receptor protein. The monoclonal antibody will specifically bind and react with an epitope present within the transactivation function, A/B domain of the human estrogen receptor protein. The functional status of the A/B domain is determined by identifying and discriminating among the 8S, 4S, 5S isoforms of the A/B region as these exist in the human estrogen receptor protein.

FIELD OF THE INVENTION 
The present invention is concerned with the development of site-specific 
monoclonal antibodies raised against preselected functional domains and 
subdomains within estrogen receptor protein; and is particularly directed 
to the use of specific monoclonal antibodies directed against individual 
epitopes and amino acid sequences in the A/B domain of human estrogen 
receptor protein as a means for evaluating the functionality of estrogen 
receptors. 
BACKGROUND OF THE INVENTION 
Breast cancer is the second leading cause of death in women in the United 
States and is a major health problem. It is estimated that 180,000 new 
cases will be diagnosed each year, among whom 46,000 deaths will occur. 
Following removal of the tumor, a number of therapeutic modalities are 
used presently in management of breast cancer patients, including adjuvant 
chemotherapy, radiation therapy, endocrine therapy or a combination of 
such treatments. These treatments have been shown to prolong disease-free 
survival (DFS) and overall survival of breast cancer patients. 
Estrogen receptors (ER) are detected in 50-85% of all breast tumors, a 
positive ER identifying those patients that have a higher probability of 
responding to endocrine manipulations. A positive ER content, coupled with 
presence of progesterone receptors (PR), correlates well with the response 
rate to hormone modulation. A positive ER also correlates well with tumor 
histologic differentiation and nuclear grade, but only moderately with 
S-phase fraction, ploidy and proliferative index. 
There is a long recognized and continuing need to identify patients with a 
higher risk of tumor recurrence in order to improve the management of 
breast cancer patients. Several prognostic factors are currently used 
including clinical stage, tumor size, tumor histopathology and nuclear 
grade, marker of angiogenesis (factor VIII), tumor ploidy and 
proliferative rate, and ER and PR content. ER content, coupled to PR 
levels, is thought to provide a useful prognostic index in patients with 
metastatic tumors. However, in spite of this correlation, only about 65% 
of all patients with ER+ tumors respond to endocrine treatment. 
It is useful therefore to understand the role of estrogen receptor protein 
as such. Note that the presence of intracellular ER provides and accounts 
for both cell proliferation and new protein synthesis by estrogen 
dependent cells. The estrogen receptor in the absence of the estrogen 
hormone is biologically inactive both in vivo and in vitro; and, if the 
cells or tissues are homogenized and fractionated into cytosol and nuclear 
fractions, the estrogen receptor is found as a soluble protein in the 
cytosol. 
Although the precise mediators and reactions remain poorly understood, the 
generally accepted mechanism of action and sequence of events for ER 
interactions is believed to be as follows: When an estrogen (such as 
estradiol) is introduced to the target cells and tissues, there is 
specific binding between the estrogen and the ER protein which results in 
the formation of an estrogen/receptor protein complex. Also, at a time 
subsequent to such hormone binding, a process termed "activation and/or 
transformation" ensues which leads to the formation of functional 
estrogen/hormone receptor complexes having a high affinity for the nuclear 
components, the DNA, of the target cell. Once the hormone-receptor protein 
complex is physically formed, it is thought to translocate as a complex 
into the nucleus of the cell where it binds to the chromatin at specific 
binding sites on the chromosomes and initiates messenger ribonucleic acid 
(mRNA) transcription. New mRNA is then synthesized, chemically modified, 
and exported from the nucleus into the cytoplasm of the cell where 
ribosomes translate the mRNA into new proteins. This is the well 
recognized "estrogenic effect" on the cell--that is, the initiation of new 
protein synthesis and concomitant new cell growth/proliferation. The 
theoretical premise and the generally accepted, though poorly understood, 
mechanism of action regarding estrogen and estrogen receptor proteins and 
their interactions are described in greater detail by the following 
publications which are merely representative of the ongoing investigations 
in this field. These are: Mestre et al, Exp. Cell. Res. 81:447-452 (1973); 
King and Greene, Nature 307:745-747 (1984); Welshons et al., Nature 
307:747-749 (1984); Gorski and Gannon, Annu. Rev. Physiol. 38:425-450 
(1976); Gorski et al., Recent Prog. Horm. Res. 24:45-72 (1968); Jordan, 
V., Pharmacological Reviews 36:245-276 (1984). 
In order to truly appreciate the significance and value of the present 
invention, it is useful to summarize the major details regarding the 
different structural isoforms and functional states for the intracellular 
protein known as estrogen receptor. In the unbound state and in the 
absence of an estrogen, the estrogen receptor protein can be located in 
vitro within the cytosol and is a single protein composed of 595 amino 
acids. The molecular weight of ER protein determined from gel 
electrophoresis and other physical methods is approximately 67,000 
daltons. In soluble systems and under set test conditions, the ER protein 
can be found in alternative molecular forms or isoforms which sediment at 
either 8S, 5S, or 4S as determined by sucrose density gradient analysis. 
The 8S isoform of ER is believed to be the unactivated, untransformed form 
of the ER protein and is associated with the unbound, inactive state of 
estrogen receptor in the absence of estrogens. The 8S ER isoform is a 
large molecular weight complex, presumably associated with heat shock 
proteins, that does not bind efficiently to nuclei or DNA in vitro and is 
stabilized as a macromolecule by sodium molybdate. 
In comparison, the 4S ER isoform is a monomeric protein molecule that can 
be generated from the 8S isoform in vitro by treatment with high ionic 
strength buffers or by increasing salt concentrations (KCl or NaCl). The 
4S ER isoform binds to both nuclei and DNA-cellulose in vitro; it is 
generally termed the "activated but untransformed" estrogen receptor 
protein. From the published reports, it appears that the dissociation of 
the 8S ER isoform into the 4S isoform initiates either a major change in 
the sterochemical conformation of the protein or a direct exposure of the 
previously hidden or obscured DNA binding domain of the molecule. 
Alternatively, the 5S isoform of ER is a dimeric protein molecule created 
by the conversion of the 4S ER protein via a bimolecular reaction which is 
facilitated by elevated temperatures and/or dilution after KCl activation. 
The 5S form of ER can be generated in vitro by incubation of either the 8S 
or 4S isoforms at 28.degree.-30.degree. for 30-45 minutes in the absence 
of transformation inhibitors. It is generally believed that the 5S isoform 
of ER is both "activated and transformed" and therefore is the 
biologically active entity which binds to the DNA within the nuclei. 
Moreover, it is also this 5S isoform which is found associated within the 
nuclei subsequent to the administration of estradiol in vivo. 
It will be appreciated that the present state of knowledge regarding the 
various forms of estrogen receptor protein have been obtained and 
characterized via many different investigations, many of which employ 
physical an chemical structural and isoform analysis. Merely 
representative of such investigations and reports are the following: 
Muller et al., Endocrinology 116:337-345 (1985); Muller et al., J. Biol. 
Chem. 258:11582-11589 (1983); Endocrinology of the Breast: Basic and 
Clinical Aspects, Volume 464, Annals Of the New York Academy of Sciences, 
pages 202-217, 1986; Parmer et al., J. Steroid Biochem. 31:359-364 (1988); 
Traish et al., J. Biol. Chem. 255:4068-4072 (1980); and Muller et al., J. 
Biol. Chem. 257:1295-1300 (1982). 
Recent reports on the cloning of the complementary DNA for steroid 
receptors also have generated new information concerning the functional 
domains of steroid receptors. Functional studies have delineated the 
putative roles of several domains; and these studies have demonstrated 
that the estrogen receptor (ER) consists of multiple functional domains 
which provide and are responsible for the characteristic biological and 
physiological properties individually. The complementary DNA (cDNA) of 
human estrogen receptor also has lead directly to the elucidation of the 
human ER protein primary amino acid sequence. 
The multiple functional domains of human ER protein provide six individual 
and distinctive regions, each of which provides different properties and 
characteristics. Each of the six functional domains have been designated 
as regions "A-E" respectively. The N-terminal domain comprising regions A 
and B together span the first 184 amino acids of ER proteins; and have yet 
to be assigned a precise function in gene expression, although it is 
postulated that they are required for full functional activity in certain 
types of cells or for interaction with specific kinds of genes. 
Specifically, the A/B region or domain contains a transactivation subdomain 
(TAF-1) and is thought to be the amino acid segment important for gene and 
cell specificity. Mutations in the A/B domain have been shown to impair 
expression of ER responsive genes. Thus, receptors with an altered or 
modified A/B region can fail to produce any biological function or mass 
produce an abnormal function. 
The third functional domain is region C which encompasses the amino acid 
segment 185-263 of human ER and is necessary for the binding of ER to 
genomic DNA to occur. In particular, the DNA-binding domain is also 
involved in nuclear translocation, dimerization and transactivation; and 
it has been shown that deletion of DNA-binding region, or mutations in the 
dimerization domain or the transactivation domains eliminated ER function. 
Thus, any truncation or mutation of this C domain will lead to inactive 
(nonfunctional) ER. 
Less is known presently about the other three domains of ER protein. Region 
D (position Nos. 264-302) is believed to be the hinge area of the protein 
with as yet an undefined function. It has been shown that alterations in 
this region did not affect ER function. 
Region E (position Nos. 303-552) is believed to be the steroid binding 
domain because this region comprises an amino acid sequence which is 
generally shared between different classes of receptors for steroids. 
Region F (position Nos. 553-595) has yet to be assigned a specific 
function. 
It is important to note that regions C and E are said to be conserved among 
all the steroid receptor family members throughout the different animal 
and human classes--thereby indicating that these particular domains or 
regions are deemed to be critical for hormone receptor function generally 
within steroids as a family. Specific publications describing these 
investigations, data, and conclusions in greater detail are represented by 
the following: Kumar et al., Cell 51:941-951 (1987); Hill et al., Cancer 
Res. 49:145-148 (1989); Greene et al., Nature 320:134-139 (1986); and 
Greene et al., Science 231:1150-1153 (1986). 
It is also essential to understand the relationship between domain or 
region and structure and biological function; and to realize that 
structural alteration(s) in ER may interfere with normal ER activity by 
reduction or inhibition of characteristic biological functions. 
Alternatively, an altered ER may be constitutively active in transacting 
of E.sub.2 -responsive genes. Several studies have reported the presence 
of altered/nonfunctional ER in human breast tumors. A number of ER 
variants (ERVs) in breast tumors have been described, including those with 
base-pair insertions, deletions and transitions or those with mutations in 
DNA-binding C domain or in the hormone-binding E domain. These ERVs may 
have intrinsically impaired function or may influence ER function through 
positive or negative dominance, contributing to hormone insensitivity. 
Functional studies have also shown that deletion of the DNA-binding region 
or mutations in the dimerization and transactivation domains eliminates ER 
function without affecting E.sub.2 -binding activity. Alteration of ER 
DNA-binding activity in breast tumors and in the T47D breast cancer cell 
line have recently been described. Wang and Miksicek have reported the 
isolation of two ER mRNA variants in T47D human breast cancer cells which 
have deletions in the DNA-binding domain. These ERVs inhibit 
estrogen-dependent transcriptional activation by wild-type ER in a 
negative-dominant fashion. 
Also Faqua et al. Cancer Res. 51:105-109 (1991)! have described an ERV in 
breast tumor missing the hormone-binding region which exhibits positive 
dominance and may explain the continued expression of PR in some 
ER-tumors. Another variant expressed in ER+/PR- tumors exhibits 
negative-dominance. Katzenellenbogen et al. Programs+Abstracts Endo. 
Soc., p. 289, No. 953, 1993! have shown that three ER mutants, generated 
by random chemical mutagenesis, interfere with normal ER function. 
Clinically, the functional role of ER and the hormonal treatment of ER+ 
tumors is far less known or understood. The use of estrogens such as 
estradiol in hormonal therapy fails in about 35% of patients with ER+ 
tumors. The lack of response to hormonal manipulations has been 
attributed, at least in part, to: (a) the presence of nonfunctional ER as 
determined by its inability to recognize and bind to specific 
DNA-responsive elements and/or its inability to recruit other 
transcriptional activation factors; (b) tumor heterogeneity in which some 
tumor cells may contain functional ER while other cells may contain either 
dysfunctional ER or do not express ER at all and may become autonomous 
with respect to their hormone sensitivity, allowing tumor progression; 
and/or (c) mutations of specific estrogen-responsive genes, thus affecting 
gene expression. 
In addition, several tumor phenotypes have been described based on ER and 
PR content; and the ERVs described above influence the behavior and 
hormone sensitivity of these tumors. For example, ER+/PR+ tumors may 
express both functional and nonfunctional ER, which could serve to explain 
incomplete sensitivity to endocrine manipulations. Also, ER+/PR- tumors 
may have ER with altered DNA-binding or transactivation domains and, thus 
fail to transactivate E.sub.2 -responsive genes; ER-/PR+ tumors may have 
ER with an altered hormone-binding domain but remain constitutively 
active; and ER-/PR- tumors may not express ER or be defective in one or 
more domains and, consequently, may be transcriptionally inactive. 
Via these many different published reports, it will be noted and 
appreciated also that currently available diagnostic tests to determine 
the presence or absence of ER protein in tumors (and the many assays 
employed for research purposes regarding ER protein) depend and rely 
either on binding of ligand to the receptor protein in cell extract or 
upon the existence of specific antibodies raised against ER. A variety of 
different polyclonal antisera have been prepared against estrogen receptor 
protein and against the nuclear binding estradiol-receptor complex 
typically identified as "estrophilin" Raam et al., Mol. Immunol. 
18:143-156 (1981); Greene et al., J. Ster. Biochem. 11:333-341 (1979); 
Greene et al., Proc. Natl. Acad. Sci. USA 74:3681-3685 (1977)!. Similarly, 
a large number of monoclonal antibodies against human and animal estrogen 
receptor proteins and estrophilins have been prepared for many different 
investigational purposes Greene et al., Proc. Natl. Acad. Sci. USA 
74:3681 (1977); Green et al., Proc. Natl. Acad. Sci. USA 77:157-161 
(1980); Greene et al., Proc. Natl. Acad. Sci. USA 77:5515 (1980); Brogna 
et al., Biochem. 23:2162-2168 (1984); Fauque et al., J. Biol. Chem. 
260:15547-15553 (1985); and Moncharmont et al., Biochemistry 23:3907-3912 
(1984)!. 
The common flaw and recurring problem of these known polyclonal and 
monoclonal antibodies against ER is their uniform and consistent failure 
to be site specific. This failure, in turn, produces erroneous empirical 
results and unreliable information--not only for investigational purposes 
but also in clinical applications of such antibodies for 
diagnostic/therapeutic purposes. As a major example, the measurement of 
estrogen receptors in human breast carcinomas has been the primary tool 
and favored diagnostic method for choosing between hormonal and cytotoxic 
chemotherapy when treating breast cancer patients. A variety of different 
immunoassays employing anti-ER antibodies are presently known and used for 
this purpose. These are examplified by the following publications: U.S. 
Pat. Nos. 4,232,001; 4,293,536; 4,215,102; and 4,711,856. See also 
European Patent Application Publication No. A2-0129669 published Jan. 2, 
1985. Unfortunately, the immunoassays employing conventionally obtained 
monoclonal antibodies for these measurements have been found to be 
frequently unreliable and often non-specific. The nature and variety of 
problems of these unreliable and non-specific monoclonal antibodies are 
illustrated by the following publications: Raam, S. and D. M. Vrabel, 
Clin. Chem. 32:1496-1502 (1986); Raam, S. and D. M. Vrabel, Clin. Chem. 
34:2053-2057 (1988); Raam, S., Steroids 47:337-340 (1986); and Raam, S., 
Clin. Chem. 22:1107-1108 (1987). Clearly, therefore, given all the 
presently known antibodies, assays, and immunological techniques, one 
still cannot accurately predict which of the estrogen receptor positive 
tumors will respond to hormonal treatment. 
The causes of the present dilemma are in fact two-fold: First is the 
failure of the monoclonal antibodies and polyclonal antisera to be 
sufficiently site-specific in order to demonstrate the presence of 
estrogen receptor in its various multiple forms. Second is the failure (in 
so far as is presently known) of these conventionally known antibodies to 
differentiate between functional and nonfunctional ER. 
Currently, human breast tumor estrogen receptor (ER) values are used as 
prognostic factors in determining treatment of breast cancer patients with 
tamoxifen. ER content in tumor tissue is usually determined by either 
radioreceptor assays or immunochemical techniques. Binding of labeled 
estrogen hormone (ligand) to ER does not provide sufficient information 
concerning the structural or functional integrity of the DNA-binding 
domain or the transactivation function located in the A/B region of the 
receptor. Similarly, immunochemical or immunocytochemical analysis, in 
soluble fractions or in fixed tissue sections, using antibodies with 
undefined binding epitopes do not provide information on the functional 
domains of ER. 
It is now clearly apparent to practitioners and clinicians ordinarily 
skilled in this art that so long as these insufficiently specific 
antibodies remain in clinical use, many repetitive failures in the known 
immunoassay systems will occur; and the ability to identify that 
proportion of breast cancer patients which would be sensitive and 
responsive to estrogen hormonal treatment will remain plagued with 
uncertainty and inaccuracy. For these reasons, the development of 
site-specific antibodies common to the transactivating A/B domain of ER 
and their use within conventionally known diagnostic immunoassays would 
therefore be recognized generally as a major advance and fundamental 
improvement in antibody materials, assay reliability, and therapeutic 
benefit. 
SUMMARY OF THE INVENTION 
The present invention has multiple aspects and parts which are intimately 
related. 
A first aspect of the invention provides a monoclonal antibody specific for 
epitope within amino acid residues 1-184 of an estrogen receptor protein, 
said monoclonal antibody having binding specificity for a single epitope 
within the A/B domains in the activated but untransformed (4S) forms and 
in the activated and transformed (5S) forms of estrogen receptor protein 
but which does not bind with native (8S) forms of estrogen receptor 
protein. 
A second aspect of the present invention provides a hybridoma which 
produces a monoclonal antibody specific for an epitope within amino acid 
residues 1-184 of an estrogen receptor protein, said hybridoma comprising: 
an antibody producing cell producing a monoclonal antibody having binding 
specificity for a single epitope within the A/B domains in the activated 
but untransformed (4S) forms and in the activated and transformed (5S) 
forms of estrogen receptor protein but which does not bind with native 
(8S) forms of estrogen receptor protein; and 
a tumor cell fused with said antibody producing cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention as a whole is based upon the unique approach of 
developing site-specific polyclonal and monoclonal antibodies against the 
transactivating A/B domains of estrogen receptor protein. The polyclonal 
antisera and the monoclonal antibodies are used to identify the presence 
or absence of an intact transactivating A/B domain within the ER sample 
under test; and to differentiate between functional and nonfunctional 
states of estrogen receptor in vitro. On this basis the user is able to 
determine whether the A/B domain of the ER is structurally intact and 
present in a functional or non-functional state. The invention, therefore, 
includes specifically prepared immunogens; polyclonal antisera and 
epitope-selective monoclonal antibodies which bind specifically to the A/B 
domain; and immunoassays employing these site-specific antibodies with 
cellular samples on a functional and correlative test basis. 
It will be noted and appreciated that the present invention overcomes 
several major impediments and problems normally encountered in the 
production and use of specific antibodies. These benefits and advantages 
include: 
1. Since oligopeptides of known amino acid sequence are employed as the 
hapten within the prepared immunogen, there is no longer any need for 
purification of ER, particularly human ER, for immunization purposes; thus 
a task which normally requires large resources and considerable amounts of 
tissue is eliminated. 
2. The present invention allows the user to select the precise domain 
and/or subdomains intended to be the specific binding site for the 
antibody about to be produced. Previous approaches and techniques utilized 
either the whole estrogen receptor protein or the complete hormone/receptor 
complex as the immunogen--neither of which provides any site specificity 
properties or capabilities whatsoever. 
3. The present invention precludes the binding of the site-specific 
monoclonal antibody to an ER protein when existing in an altered, 
non-functional state and is unable to undergo activation/transformation. 
To fully appreciate the present invention, it is useful to focus on a 
single kind of estrogen receptor protein, recognizing that the 
characterization of the one ER may properly be extended to include all 
other types and sources of ER protein generally. For this reason, the 
remainder of this description and the experiments and empirical data which 
follow hereinafter are limited to the use of human estrogen receptor 
(hereinafter "hER"). For this purpose also, all the presently available 
information regarding the primary amino sequence and the various 
functional domains of human estrogen receptor protein have been employed. 
Nevertheless, it will be clearly and explicitly understood, that the scope 
of the present invention encompasses the ability to prepare site-specific 
antibodies against individual domains and subdomains of ER from all 
presently known sources and origins, whether human or animal. 
I. GENERAL PREATION, REAGENTS, REACTIVE MIXTURES, AND TESTING PROCEDURES 
A detailed general description follows disclosing the means and manner 
generally of preparing oligopeptides, immunogens, immunization practices, 
preparation of hybridomas, and the isolation of epitope-specific 
monoclonal antibodies. These procedures recite and delineate the 
compositions and methods by which the ordinarily skilled practitioner in 
this art can make and use the antibodies of the present invention to 
advantage, without incurring substantive difficulties. 
OLIGOPEPTIDES 
As regards the domains of human ER protein and its different functional 
states, it will be recognized that the human receptor molecule is a single 
polypeptide having 595 amino acids in sequence in which the A and B regions 
include the amino acid segment from about positions 1-184 respectively. 
These domains are a critical region of the human ER; the A/B regions 
provides the transactivating capability in vivo for the entire ER 
molecule. Therefore, each domain individually (A or B region alone) or 
collectively (A/B together) can provide at least a portion or fraction of 
the amino acids useful as fused oligopeptides which can then serve as 
haptens for raising site-specific antibodies in accordance with the 
present invention. 
As described in detail hereinafter, oligopeptides having known amino acid 
sequences identical to chosen segments within positions 1-184 of human 
estrogen receptor ("hER") were prepared and purified. These individual 
oligopeptides were first linked to an antigenic protein carrier (such as 
keyhole limpet hemocyanin); and then were used as prepared immunogens to 
produce antibodies to the DNA-binding domain specifically. For purposes of 
practicing the present invention, however, it is not necessary that all 
these specific hER amino acid residue sequences be employed when preparing 
the immunogen. To the contrary, for human purposes, it is required only 
that an immunogenic oligopeptide be chosen whose amino acid residue 
sequence corresponds to at least one epitope commonly present within the 
entire 1-184 amino acid residues which encompass the A and B regions of 
hER. Any amino acid segment able to function as a hapten and thereby 
provide one specific epitope or site-specific binding capability for the 
resulting antibody is thus suitable for use. Moreover, although the 
characterized oligopeptides average approximately 15-30 amino acid 
residues in length, there is no requirement that any polypeptide sequence 
conform to this particular length or size for purposes of preparing the 
immunogen. In general, however, it is desirable that the oligopeptide 
representative of an ER subdomain be at least about 5-7 amino acid 
residues in number as the minimum segment size able to serve as a hapten. 
Nevertheless, any number of amino acid residues in sequence which is or 
represents a specific epitope of these domains and is able to provide the 
necessary hapten attributes and site-specificity for the prepared 
immunogen is suitable and deemed to be within the scope of the present 
invention. 
In addition, although it is desirable that the amino acid residue sequence 
of the chosen oligopeptide be in fact identical to the true, naturally 
occurring amino acid sequences of the A/B region, this is not an absolute 
requirement. To the contrary, it is expected that substantial variation of 
the amino acid residues which differ from those normally found within the 
A/B regions of the hER molecule is permissible; nevertheless, it is most 
desirable for the chosen amino acid sequence to be completely or very 
nearly identical to the sequences normally existing in the A/B domains, 
particularly when the oligopeptide is of a minimal length and size. 
Synthesis Of Oligopeptides 
The oligopeptides employed herein were prepared using conventionally known 
solid phase peptide synthesis methods Merrifield, R. B., J. Am. Chem. 
Soc. 85:2149 (1963)!. Once synthesized, the individual oligopeptides were 
purified by gel filtration and analyzed for purity by HPLC. Analysis of 
the amino acid composition correlated well with the primary sequence. Each 
peptide contained one .sup.3 H!-labeled amino acid as a tracer. This 
provided the means for determining the efficiency of coupling to the 
various carrier proteins. 
Reagents 
Isotopes and Chemicals 
6,7.sup.3 H! estradiol (40-60) Ci/mmol) (.sup.3 H!E.sub.2), 7.alpha., 17 
.alpha.-dimethyl 17.alpha.-methyl .sup.3 H!-19-nortestosterone (70-85 
Ci/mmol) (.sup.3 H! DMNT) 16 .alpha.-ethyl-21-hydroxy-19-nor 6,7.sup.3 
H!-pregn-4-ene-3,20-dione (40-60 Ci/mmol) (.sup.3 H! ORG 2058), .sup.3 
H!-triamcinolone acetonide (20-40 Ci/mmol) (.sup.3 H!TA), unlabeled DMNT, 
and ORG 2058 were obtained from Amersham, Arlington Heights, Ill. 
Unlabeled TA was obtained from Sigma Chemical Co., St. Louis, Mo., 
unlabeled diethylstilbestrol (DES) and E.sub.2 were obtained from 
Steroids, Wilton, N.H. All other chemicals were reagent grade and were 
obtained from commercial sources. 
Buffers and Solutions 
Buffer TGT: 50 mM Tris-HCl, 10% glycerol, 10 mM thioglycerol (pH 7.4 at 
2.degree. C.). 
Buffer TGT/MO: buffer TGT with 10 mM sodium molybdate. 
Buffer TT: 50 mM Tris-HCl, 10 mM thiolglycerol (pH 7.4 at 2.degree. C.). 
Buffer TT/KCl: buffer TT containing 0.4M KCl. 
IMMUNOGENS 
After an oligopeptide has been synthesized, it is then chemically coupled 
to a protein carrier to form the prepared immunogen. The suitable protein 
carriers available for this purpose are conventionally known and available 
in great variety from diverse sources. The only requirements regarding the 
characteristics and properties of the carrier protein are: First, that the 
carrier be in fact antigenic alone or in combination with the chosen 
oligopeptide; and second, that the carrier protein be able to present the 
chemically bound oligopeptide after administration in vivo such that 
antibodies specific against at least one epitope present concurrently or 
shared between the A and/or B domains of the ER molecule are produced. 
Clearly, as in the experiments described hereinafter, the preferred choice 
of protein carrier for immunization purposes was keyhole limpet hemocyanin 
(hereinafter "KLH"). However, any other carrier protein compatible with 
the host to be immunized is also suitable for use. Examples of such other 
carrier proteins include bovine serum albumin, gelatin, thyroglobulin, and 
the like. 
Coupling Of Oligopeptides To Carrier Proteins 
To prepare effective immunogens, the oligopeptides were individually 
coupled to known carrier proteins to form antigenic immunogens. The 
carrier proteins of choice were keyhole limpet hemocyanin and bovine serum 
albumin (hereinafter "BSA"). The KLH-coupled peptides were used as 
immunogens while the BSA-coupled peptides were used only for screening 
assays. This coupling procedure was performed as follows. 
KLH and BSA were dissolved in phosphate buffered saline (PBS: 0.2 g 
KH.sub.2 PO.sub.4, 8 g NaCl, 2.16 g Na.sub.2 HPO.sub.4 7H.sub.2 O in one 
liter of distilled water, pH 7.5) to give a final concentration of 1 
mg/ml. One hundred mg of each peptide was then dissolved in 10 ml of KLH 
solution and 50 mg of each peptide was dissolved in 5 ml of BSA solution. 
The pH of the mixtures was adjusted to 9 with 0.1M LiOH. The coupling of 
the peptides to the carrier proteins was initiated by dropwise addition of 
6.25% glutaraldehyde to achieve a final concentration of 1% glutaraldehyde. 
Each mixture was then incubated at 0.degree.-4.degree. C. for 1 hour with 
gentle agitation. Aliquots (50-200 ul) were then removed and used to 
determine total peptide concentration by radioactivity counting. The 
remainder of each mixture was then transferred to dialysis tubes and 
dialyzed extensively against four changes of PBS. Aliquots were then 
removed after dialysis and counted to determine the efficiency of 
coupling. The remaining dialyzed material was divided into 1 m. aliquots 
and frozen at -80.degree. C. until needed. 
Immunization Procedures 
All immunizations and immunization procedures are performed in the 
conventionally known manner. It is expected that under certain use 
conditions, adjuvants will be employed in combination with the prepared 
immunogens. Alternatively, the prepared immunogens may be used alone and 
administered to the host in any manner which will initiate the production 
of specific antibodies. 
In addition, the harvesting of polyclonal antiserum and the isolation of 
antibody containing sera or antibody producing cells follows the 
conventionally known techniques and processes for this purpose. Similarly, 
the preparation of hybridomas follows the best practices developed over 
recent years for this specific purpose Marshak-Rothstein et al., J. 
Immunl. 122:2491 (1979)!. A complete detailed description of the preferred 
techniques and procedures follows hereinafter. 
Immunization Of Rabbits 
New Zealand white female rabbits (7-9 lbs) were used. Prior to 
immunization, serum was collected from each rabbit by bleeding through the 
ear artery and designated as preimmune serum. One day later each animal was 
injected subcutaneously at multiple sites along the back with a total of 1 
ml of an emulsion made by mixing equal volumes of complete Freund's 
adjuvant and KLH-conjugated peptide mixture. The final emulsion contained 
1 mg/ml of the desired peptide. After three weeks the rabbits were boosted 
with the antigen in incomplete Freund's adjuvant. Two weeks after the 
booster shots, the rabbits were bled and the sera collected and tested for 
the presence of peptide-specific antibodies by enzyme-linked 
immunoabsorbent assay (ELISA). The animals were then given booster shots 
several times and bled 14 days after the final booster. This procedure 
provided all the peptide-specific polyclonal antisera described 
hereinafter. 
Immunization Of Mice 
In addition, groups of female mice (BALB/cA/J F.sub.1 ! 6-8 weeks old were 
also immunized by injecting s.c. 100 ug of the designated oligopeptides, 
emulsified in Freund's complete adjuvant. Two s.c. booster injections were 
given at 3 week intervals. The mice were bled through the vein and the sera 
were tested for antibodies by sucrose density gradient analysis. Those mice 
which appeared to have antibodies against ER were then selected. After one 
month of rest the mice were given 100 ug of the antigen in PBS 
intraperitoneally (i.p.) and sacrificed three days later; then their 
spleens were removed and used for cell fusion and the production of 
monoclonal antibodies. 
ANTIBODIES 
Preparation Of Hybridomas And Isolation Of Monoclonal Antibodies 
Cell fusion was carried out by the method of Marshak-Rothstein et al. J. 
Immunol. 122:2491 (1979)!. Briefly, mouse spleens were excised; the fat 
and mesenteric tissues were removed quickly; and a single cell suspension 
was made by squeezing the spleen between two glass slides in Hank's 
balanced salt solution (HBSS) buffered with 0.02M phosphate, pH 7.2. Red 
blood cells were lysed by brief incubation in ammonium chloride lysis 
buffer. Spleen cells (5.times.10.sup.7 cells) were mixed with Sp 2/0 cells 
(5.times.10.sup.6 cells) in round bottom tubes and pelleted at 700.times.g 
for 5 min at 22.degree. C. The cells were resuspended in serum-free DME 
and centrifuged. After removal of the supernatant, the cell pellet was 
resuspended for six minutes in 0.5 ml of polyethylene glycol 1,500 ("PEG", 
30% v/v) Baker Chemical Co., Phillipsburg, N.J.), followed by addition of 4 
ml of serum free DMEM (Dulbecco's Modified Eagle's Medium) to dilute out 
the PEG. The cell suspensions were transferred into petri dishes 
(100.times.17 mm) and DME containing 20% FCS (fetal calf serum) was added 
and the cultures were kept at 32.degree. C. for 24 h under 5.6% CO.sub.2. 
The cells were then pelleted and resuspended in HAT (hypoxanthine, 
aminopterin, and thymidine) conditioned medium (1.times.10.sup.6 
cells/ml). Aliquots of the cells suspension (0.1 ml) were dispensed into 
96-well flat bottom microtiter dishes and incubated at 37.degree. C. Seven 
days later the hybridoma cells were treated with 0.1 ml of conditioned 
media (DME, HT). After another two days, the resulting hybridomas were 
screened by enzyme-linked immunoabsorbent assay (ELISA) against 
ESA-conjugated oligopeptides. 
The isotype of each monoclonal antibody was determined by ELISA. Microtiter 
plates coated with the immunogenic oligopeptide were incubated with 
aliquots of the spent media from the hybridoma. Bound antibody heavy chain 
class was determined by addition of goat-antimouse isotyping reagents 
(Southern Biotech, Birmingham, Ala.) diluted 1:1,000 in PBS-0.2% BSA, 
followed by alkaline phosphatase conjugated rabbit-antigoat secondary 
antibody and the substrate. 
Hybridoma clones that tested positive by ELISA were recloned by limiting 
dilution. Cells were diluted to 1, 0.3, 0.1 cell equivalent/ml in DMEM 
containing 2% FCS and BALB/c peritoneal exudate cells (5.times.10.sup.4 
cells ml) and then plated in 96-well microtiter plates. After ten days, 
wells with single hybridoma clones were identified by microscopic 
examinations and tested for presence of antibodies by ELISA. Clones that 
tested positive were expanded in large flasks, spent media were collected, 
and cells were either used for ascites productions or frozen for later use. 
Polyclonal Antisera and Monoclonal Antibodies 
Once obtained from their living hosts, the polyclonal antisera and the 
monoclonal antibodies were evaluated and verified for their ability to 
bind specifically with the A and B domains of the ER. The polyclonal 
antiserum prepared as described herein has been found to bind specifically 
with the A/B region of human ER in the 8S, 4S, and 5S isoforms. The 
polyclonal antisera therefore are able to identify the presence of intact 
A/B regions in the various ER states--be they the unactivated 8S form; the 
activated but untransformed 4S form; or the activated and transformed 5S 
form. When utilized within the assay procedures for this purpose, these 
polyclonal antisera will accurately detect the presence of the A/B region 
with the ER intracellularly; and will additionally provide the background 
by which to identify the functional status of the detected ER protein. 
In comparison, as empirically demonstrated hereinafter, monoclonal 
antibodies raised in the described manner are epitope-specific binding 
antibodies; and thus will bind with the activated human ER in the 
transformed or untransformed states. This corresponds to the capability of 
detecting hER in the 4S and the 5S forms only. These monoclonal antibodies 
are site-specific in their properties; and they will not and do not bind 
to the unactivated and untransformed 8S form of hER. Accordingly, the 
monoclonal antibodies will service to identify both the functional 
"activated but untransformed" as well as the "activated and transformed" 
state of the ER protein actually present within the cells or tissues being 
empirically evaluated. 
Finally, when the polyclonal antisera and the monoclonal antibodies are 
employed within immunoassays to determine the presence of ER within a 
cellular sample, a direct comparison of the empirical results obtained 
using polyclonal antisera and monoclonal antibodies provides a direct and 
unequivocal measure of the functional status and activated/transformed 
state of the ER protein being empirically detected. 
Moreover, the ability to identify not only the functional status but also 
the activation and transformation states of human estrogen receptor in a 
cellular sample thus allows the use of assay procedures for the accurate 
quantitation of cytosolic estrogen receptors in breast cancer tissue 
samples. By employing the polyclonal antisera and the monoclonal 
antibodies within individual assays, the resulting data can be used to 
correlate not only the mere presence of estrogen receptor but also the 
quantity of function in the receptor within the tissue obtained from a 
single source or patient. On this basis, it now becomes possible to 
segregate breast cancer patients more accurately into two populations: 
those who are likely to respond to hormonal therapy via the presence of an 
intact and functional estrogen receptor; and those who are not likely to 
respond to hormones despite the presence of estrogen receptors because 
these are either not intact and/or non-functional. 
PREFERRED TEST PROTOCOLS 
To demonstrate the uses of the polyclonal antisera and the monoclonal 
antibodies for such assay purposes, a set of preferred protocols are 
provided which will illustrate the range of methods and manipulative steps 
able to be employed in the performance and the utilization of immunoassays. 
It will be expressly understood however, that the procedural steps 
described hereinafter are merely representative of the nature and 
manipulative steps employed within immunoassays generally. The described 
protocols are not self-limiting and are not restrictive to only the 
described manipulative steps and the test conditions employed. To the 
contrary, it is deemed and expected that a wide variety of homogeneous and 
heterogeneous immunoassay systems may be employed; that the parameters of 
concentration, volume, temperature, and choice of reagents can be varied 
extensively at will; that the identifying labels used with the polyclonal 
antisera and the monoclonal antibodies in such assays may be either 
isotopic or non-isotopic in nature; and that the protocols might be 
embodied as kits or other test apparatus in commercially salable form 
rather than individually prepared reagents and reactants. The present 
invention presumes and incorporates by reference any conventionally known 
immunoassay techniques, procedures, protocols, substrates, and other 
non-decisive factors and parameters--all of which may be usefully employed 
within any given immunoassay procedure. None of these are deemed to be 
essential or dominant criteria when performing the methods of the present 
invention. 
Accordingly, for illustrative purposes only, preferred protocols utilizing 
polyclonal antisera and monoclonal antibodies specific against at least 
one epitope in the A/B domains of human estrogen receptor are given 
hereinafter. 
A. Estrogen Receptor Assay Protocol 
Buffers 
TEGM buffer consists of 10 mM Tris-HCl; 1 mM ethylenediamine tetracetic 
acid (EDTA); 10% vol/vol glycerol; 10 mM sodium molybdate; 10 mM 
monothioglycerol; and 0.02% sodium azide (pH 7.4 at 2.degree. C.). 
Preparation Of Cytosol Fractions 
The tissue sample is pulverized, weighed, and placed in a test tube on ice. 
Unless otherwise stated, all manipulations are carried out at 
0.degree.-4.degree. C. Ice cold TEGM buffer is added to the tissue in a 
4:1 vol/wt and homogenized with a polytron Pt-10 using 5 sec. bursts and 
30 sec. cooling periods in between bursts. The homogenate is then 
transferred into ultracentrifuge tubes; and the homogenate is centrifuged 
at 100,000.times.gravity for 45 minutes to obtain the high speed 
supernatant of cytosol. The cytosol is transferred to a clean tube and 
placed on ice. 
Preparation Of Radioactive Estradiol Stock Solutions 
.sup.3 H! estradiol is obtained in solution of benzene/ethanol. Aliquot is 
removed and dried under nitrogen. The dried material is resuspended in a 
small volume (2-5 ul) of ethanol; buffer is then added to dissolve the 
radiolabeled estradiol. The concentration of the estradiol E.sub.2 ! is 
then determined by conventional radioactive counting. 
Incubation Of The Cytosol With Radiolabeled Estradiol 
Aliquots of the cytosol are then incubated with 5 nM of radiolabeled 
estradiol at 0.degree. C. for 16 hours to form the estrogen 
receptor/estradiol complexes. To determine the nonspecific binding, 
parallel aliquots of cytosol were incubated with .sup.3 H!E.sub.2 and a 
100 fold molar excess of unlabeled diethylstilbestrol. 
Removal Of Free Radioactive Estradiol With Dextran Coated Charcoal "DCC" 
The DCC suspension in TEGM buffer is centrifuged and the supernatant is 
discarded. The cytosol incubation is then transferred into the DCC pellet 
and mixed and kept on ice for 20 minutes with intermittent mixing. The 
suspension is then centrifuged at 1,000.times.g (gravity) for 10 minutes 
and the supernatant is used as a source of labeled cytosol. 
B. Determination Of Estrogen Receptors By An Enzyme Immunoassay 
96-well Microtiter plates are preferably used. Aliquots of bovine serum 
albumin conjugated oligopeptide in 50 ul of phosphate buffered saline (3 
ug/50 ul) are pipetted into each well and allowed to bind at 0.degree. C. 
for 16 hours. The wells are then coated with 200 ul of 2% bovine serum 
albumin (BSA) in phosphate buffered saline (PBS) for 1-2 hours at 
25.degree. C. The plates are then washed with PBS three times and used for 
competitive binding assay. 
An aliquot of the desired antibody, either monoclonal or polyclonal, is 
diluted 1:2,000 with BSA/PBS and 50 ul are incubated for 16 hours at 
0.degree.-2.degree. C. with increasing volumes of the human breast tissue 
cytosol (10-200 ul). This allows the antibody receptor interaction to form 
the antigen-antibody complex. The (polyclonal or monoclonal) 
antibody/cytosol mixture is then added to the wells of the microtiter 
plate and incubated at 0.degree.-2.degree. C. for an additional 16 hours. 
The individual wells are then washed three times with PBS; and a secondary 
antibody previously conjugated to alkaline phosphatase is added in 1:5,000 
dilution in BSA/PBS to each well and the reaction mixture incubated at 
25.degree. C. for 2 hours. The unbound (polyclonal or monoclonal) antibody 
is then removed and the wells washed three times in PBS. An enzyme 
substrate for alkaline phosphatase is then added to each well and 
incubated at 25.degree. C. for 30 minutes in the dark. The color reaction 
is then stopped by addition of 0.5N NaOH. The color product of the 
reaction is measured by ELISA microtiter reading at 450 nm. 
Control incubations are made in the absence of cytosol. This allows 
measurement of all the antibody bound in the reaction mixtures. 
Nonspecific binding is determined by omitting the primary (polyclonal or 
monoclonal) antibody. Additional controls are provided by wells that were 
coated with BSA only and do not contain any oligopeptide. Calf uterine 
cytosol with known estrogen receptor content is used as external standard 
to evaluate the reproducibility of the assay. 
C. Determination Of The Functionality Of Human Estrogen Receptor 
An aliquot of the cytosol to be tested is labeled with radioactive 
estradiol as described above and is incubated with 25 ug of the antibody 
at 0.degree.-2.degree. C. for 4-16 hours in the presence of 0.4M KCl. The 
total sample is then layered together with .sup.14 C-labeled BSA and human 
gamma-globulin using a 5-20% sucrose density gradient (made in TEGM buffer 
containing 0.4M KCl, in 4 ml polyallomer ultracentrifuge tubes). The 
gradients are centrifuged at 50,000 rpm in an SW60 Beckman rotor for 18 
hours at 2.degree. C. The gradients are then fractioned into 0.1 ml 
individual fractions, 0.5 ml of water and 4 ml of Liquiscint are then 
added and the samples counted for radioactivity. The intact estrogen 
receptor binds to the antibody and sediments in the 7-8 S region of the 
gradient. Estrogen receptors with defective, missing, or altered domains 
will not interact with antibody and therefore will sediment in the 4-5 S 
region. 
D. Binding Of (Polyclonal Or Monoclonal) Anti-ER Antibodies To ER 
Sources of Tissue 
Human breast cancer tissue was obtained through the Steroid Receptor Assay 
Laboratory of Boston University. Tissue procurement was performed as 
described in Muller et al., Cancer Res. 40:2941 (1980). Calf uterine 
tissue was obtained from a local slaughter house as described previously 
Traish et al. , Endocrinology 118:1327 (1986)!. Rat uterine tissue was 
obtained from 21-23 day old female Sprague-Dawley CD rats. Rat prostates 
were obtained from mature 24 h castrated males (Charles River Breeding 
Laboratories). 
Cytosol fractions from each kind of tissue were prepared in TT buffer as 
described previously Traish et al., Endocrinology 118:1327 (1986)!. 
Briefly, fresh tissue or frozen tissue powder was homogenized (1 g/4 ml) 
in buffer TT, pH 7.4 at 2.degree. C. The homogenate was then centrifuged 
at 105,000.times.g for 45 min. at 2.degree. C. and the supernatant 
fraction (cytosol) was used for receptor binding studies. 
Labeling Of Cytosols With Steroid Hormones 
To label the ER, aliquots of the tissue cytosols were incubated at 
2.degree. C. for 4 h with 5 nM .sup.3 H!E.sub.2 in the absence (total 
binding) or presence (non-specific binding) of a 100 fold molar excess of 
unlabeled DES. Progesterone receptors were labeled by incubating calf 
uterine cytosol with 15 nM .sup.3 H!ORG2058 in the absence or presence of 
unlabeled ORG2058 as described previously Traish et al., Steroids 47:157 
(1986)!. Androgen receptors were labeled by incubating rat prostatic 
cytosol with 10 nM .sup.3 H!DMNT in the absence or presence of unlabeled 
DHT. Glucocorticoid receptors were labeled by incubating calf uterine 
cytosol with 10 nM .sup.3 H!TA and 20 nM unlabeled ORG2058 in the absence 
or presence of unlabeled TA as described previously. At the end of the 
incubation, free radioactivity was removed with DCC pellets and the 
supernatant used for antibody-receptor interactions. 
Sucrose Density Gradient Analysis 
Sucrose density gradients (8-30%) were prepared in TT buffer containing 
0.4M KCl. In some experiments (as indicated) the gradients were made 8-30% 
in TT buffer containing 10% glycerol with or without 0.4M KCl. Samples to 
be analyzed were layered on the gradient together with .sup.14 C-labeled 
sedimentation markers. The gradients were centrifuged at 50,000 rpm in an 
SW60 rotor for 18 h at .degree.C. Gradients were fractioned into 
individual 0.1 ml fractions, scintillation fluid was added, and 
radioactivity counted. 
To further document and demonstrate the individual parts of the present 
invention and the major advantages and abilities provided by the invention 
as a whole, a variety of different experiments were performed and the 
resulting data recorded. These empirical experiments and data are provided 
hereinafter in order that the properties, characteristics, uses, and 
advantages of each component part may be properly appreciated and 
understood. It will be recalled, however, that these experiments are 
directed to estrogen receptor; and that the specific embodiments, 
procedures, modes of preparation, and immunoassays performed are merely 
illustrative and representative of the totality of embodiments encompassed 
within the scope of the present invention. 
II. ANTIBODIES SPECIFIC FOR AT LEAST ONE EPITOPE WITHIN THE A/B REGION OF 
hER 
A. Preparation Of Synthetic Oligopeptides And Immunogens Representative Of 
The A/B Domains Within Human Estrogen Receptor Protein 
The A/B region of human estrogen receptor (hER) is encompassed by the amino 
acids at about positions 1-184 in the native protein. FIG. 1 and FIG. 1A 
depict the amino acid sequence of the transactivation A and B domains and 
of the three oligopeptides chosen as haptens and immunogens to obtain 
specific monoclonal antibodies and polyclonal antisera. It will be noted 
that: 
Peptide NMT-1 represents the segment of amino acid residues from positions 
140-154 within native hER as: 
EQU (140) Thr-Val-Arg-G17-Ala-Gly-Pro-Pro-Ala-Phe-Tyr-Arg-Pro-Asn-Ser (154). 
Peptide NMT-2 represents the segment of amino acid residues from positions 
155-169 within native hER as: 
EQU (155) Asp-Asn-Arg-Gln-Gly-Gly-Arg-Glu-Arg-Leu-Ala-Ser-Thr-Asn (169). 
Peptide NMT-3 represents the segment of amino acid residues from positions 
170-184 of the native hER: 
EQU (170) Asp-Lys-Gly-Ser-Met-Ala-Met-Glu-Ser-Ala-Lys-Glu-Thr-Arg-Tyr (184). 
Cumulatively, therefore, the three peptide sequences are identical to about 
22% of the total A/B domains of human ER. The conserved cystein residues 
believed to play a role in the tertiary structure of the putative 
zinc-binding fingers are noted by asterisks within FIG. 1. 
Synthesis Of Oligopeptides 
The oligopeptides NMT-1, NMT-2 and NMT-3 were prepared using conventionally 
known solid phase peptide synthesis methods Merrifield, R. B., J. Am. 
Chem. Soc. 85:2149 (1963)!. Once synthesized, the individual oligopeptides 
were purified by gel filtration and analyzed for purity by HPLC. Analysis 
of the amino acid composition correlated well with the primary sequence. 
Each peptide contained one .sup.3 H!-labeled amino acid as a tracer. This 
provided the means for determining the efficiency of coupling to the 
various carrier proteins. 
Immunization Of Mice 
Groups (four animals/group) of female mice (BALB/cA/J) F.sub.1 ! 6-8 weeks 
old were also immunized by injecting s.c. 100 ug of the designated 
oligopeptides, emulsified in Freund's complete adjuvant. Two s.c. booster 
injections were given at 3 week intervals. The mice were bled through the 
vein and the sera were tested for antibodies by sucrose density gradient 
analysis. Those mice which appeared to have antibodies against ER were 
then selected. 
Polyclonal Antisera Production 
The immunogen and the raising of antisera did show some variation among the 
host animals (6 mice per group). Positive polyclonal antisera, as 
determined by sucrose density gradient analysis were obtained against all 
peptides, however, the response rate was different for each peptide used 
as an immunogen. While all mice (6 per group) immunized with either 
peptides NMT-1 or NMT-2 produced positive polyclonal antisera, only two 
positive antisera were obtained against peptide NMT-3 from the animals 
employed. 
Within this detailed description, the individual peptide specific 
polyclonal antisera obtained against peptide NMT-1 will be referred to as 
poly-1 (A-D) since each antiserum was individually obtained from only one 
rabbit and was never mixed with any other antiserum. Similarly, polyclonal 
antisera raised against peptide NMT-2 will be identified as poly-2 (A-D) 
respectively; and polyclonal antisera specific for peptide NMT-3 are 
individually designated as poly-3 (A-D). The properties, specificity, 
titer, and other characteristics of the polyclonal antisera were then 
empirically evaluated. 
B. Properties And Characteristics Of Anti-A/B Domain Human ER Polyclonal 
Antisera 
Experimental Series 1 
Interactions Of Anti-A/B Domain Mouse Polyclonal Antisera With 8S hER 
In cell free systems the solubilized ER can be found as 8S complexes 
(unactivated, untransformed), 4S complexes (activated but untransformed), 
or 5S complexes (activated and transformed). To test if the native 8S 
receptor complex will bind with anti-A/B domain antibodies, cytosol was 
incubated with .sup.3 H!E.sub.2 in absence or presence of unlabeled DES 
as described previously herein. After removal of free steroids with DCC, 
samples were incubated with each polyclonal antiserum for 4 h at 0.degree. 
C. and then analyzed on sucrose density gradients prepared in low salt 
buffer. 
Interaction Of The Polyclonal Antisera With The 4S and 5S hER 
To evaluate further the binding of these polyclonal antibodies, the binding 
of the antisera to the activated (4S) and transformed (5S) receptor 
complexes was then examined. 
Calf uterine was prepared in TGT buffer without molybdate. The cytosol was 
incubated at 0.degree. C. for 90 min with 5 nM .sup.3 H!E.sub.2 in 
absence or presence of unlabeled DES. Samples were then incubated at 
28.degree. C. for 30 min to induce heat transformations of ER. The samples 
were placed on ice and free steroids were removed with DCC. Aliquots of 
these incubations were then mixed with the indicated antisera and kept at 
0.degree.-4.degree. C. for 4 h. Samples were then analyzed on sucrose 
density gradients containing 0.4M KCl. 
Results 
Polyclonal antisera raised against NMT-1, (spanning amino acids 140-154) 
recognized some forms of the ER and an ER-related 55 kDa protein present 
only in nuclear extracts of estrogen-target tissues. Polyclonal antisera 
raised against NMT-2 (spanning amino acids 155-169) and NMT-3 (spanning 
amino acids 170-184) individually reacted with some forms of ER receptor 
as shown by sucrose density gradients and immunoprecipitation, but not by 
western blot analysis. The antisera bound the unactivated (8S) ER, the 
salt-activated (4S), and heat transformed (5S) ER complexes. All antisera 
were found to be site-specific since binding of salt-activated ER to the 
antisera was inhibited by preincubation of the antisera with 50 ug/ml of 
free synthetic peptide. These results are summarized by Table 1 below. 
TABLE 1 
__________________________________________________________________________ 
Reactivity of Polyclonal Antisera 
Raised Against hER 
Form of hER Detected 
Polyclonal 
Animal Producing 
Oligopeptide 
Unactivated 
Salt-Activated 
Heat-Transformed 
Antiserum 
Positive Antiserum 
(Position Nos.) 
(8S) (4S) (5S) 
__________________________________________________________________________ 
poly-1 (A-D) 
4 of 4 NMT-1 (Nos. 140-154) 
Yes Yes Yes 
poly-2 (A-D) 
4 of 4 NMT-2 (Nos. 155-169) 
Yes Yes Yes 
poly-3 (A-D) 
1 of 4 NMT-3 (Nos. 170-184) 
Yes Yes Yes 
__________________________________________________________________________ 
Experimental Series 2 
Lack Of Species Specificity Of Anti-A/B Domain Mouse Polyclonal Antisera 
Raised Against hER 
Since the A/B Domain of oligopeptides were synthesized according to the 
published amino acids residue sequence of the human ER, it was important 
to determine the binding specificity of these antisera. Accordingly, 
tissue cytosols (5-7 mg protein/ml) from human breast cancer, calf uterus, 
and rat uterus were prepared and labeled with .sup.3 H!E.sub.2 as 
described previously herein. Aliquots of the DCC-treated cytosol were then 
reincubated at 0.degree. C. for 4 h with the three polyclonal antisera. 
Samples were then analyzed on sucrose density gradients. The results are 
given by Table 2 below. 
Table 2 summarizes the overall results of incubation of .sup.3 
H!E2-labeled ER from calf uterine cytosols, and rat uterine cytosols, and 
human breast cancer tissue cytosols with the three polyclonal antisera. 
All three antisera recognized and bound to ER from the various animal 
species, albeit, with less affinity for the rat uterine ER. Clearly there 
is an absence of species specificity for these anti-A/B domain antisera. 
TABLE 2 
______________________________________ 
Reaction With Differing Cytosols of ER: 
Polyclonal Antisera 
Human Breast Cancer 
Rat Uterus 
Calf Uterus 
______________________________________ 
poly-1 (A-D) 
Yes Yes Yes 
poly-2 (A-D) 
Yes Yes Yes 
poly-3 (A-D) 
Yes Yes Yes 
______________________________________ 
This lack of species specificity for the A/B domain is also shown by FIGS. 
2-4 respectively--which demonstrate the absence of specificity for 
anti-(NMT-1 ), anti (NMT-2), and anti-(NMT-3) mouse polyclonal antisera 
individually. 
FIGS. 2A-2D illustrate the binding of four different anti-NMT-1 mouse 
polyclonal antisera to estrogen receptor in calf uteri (CU-ER). Calf 
uterine cytosol was prepared in buffer TEGT and labeled with 10 nM .sup.3 
H!ER for 2 hours at 0.degree. C. either with or without unlabeled DES to 
determine the nonspecific binding. At the end of the incubation, samples 
were treated with DCC at 0.degree. C. for 30 min. After centrifugation, 
aliquots of the supernatant (200 ul) were further incubated with the 
preimmune sera (open circles) as a control or with one of four individual 
antisera (closed circles) for 4 hours at 0.degree. C. and then analyzed on 
SDG/0.4M KCl. Each of FIGS. 2A-2D represents a different anti-NMT-1 
antiserum. 
Similarly, FIGS. 3A-3D illustrate the binding of four different anti-NMT-2 
mouse polyclonal antisera to Cu-ER. Experimental conditions and protocols 
are identical to those for FIG. 2. FIGS. 3A-3D represent the antisera of 
four individually immunized mice. Arrows represent the ovalbumin marker. 
Finally, FIGS. 4A-4D illustrate the binding of four different anti-NMT-3 
mouse polyclonal antisera to CU-ER. Experimental conditions and protocols 
are identical to those for FIGS. 2 and 3. FIGS. 4A-4D represent the 
antisera of four different immunized mice. Arrows represent the ovalbumin 
marker. 
C. Anti-A/B Domain Specific Monoclonal Antibodies 
The Choice Of Mouse Antisera Prepared Against Oligopeptide NMT-1 
The polyclonal antibodies to peptide NMT-1 were shown to cross-react with a 
nuclear protein that was present mainly in ER+ breast tumors. For this 
reason, monoclonals to peptide NMT-1 were obtained. five clones were 
isolated but only two were characterized, NMT-1-C6 and NMT-1-E7. These 
were characterized with respect to their ability to recognize the native 
ER. 
Experimental Series 3 
ELISA Assay Of Monoclonal Antibodies Prepared Against Peptide NMT-1 
After cell fusion, the tissue culture supernatants from the various clones 
were initially screened for monoclonal antibodies (MAb) against 
oligopeptide NMT-1 conjugated to BSA. Fifteen clones appeared to contain 
MAbs against this oligopeptide. To determine if the clones secreted 
immunoglobulins against the oligopeptide, the spent tissue culture medium 
from each clone was assayed by ELISA for presence of antibodies. The assay 
was performed as follows. 
Aliquots (50 ul) of the bovine serum albumin conjugated peptide (3 ug) in 
PBS were dispersed into the microtiter wells and incubated at 
0.degree.-4.degree. C. for 16-20 h. The plates were then blocked with 2% 
BSA in PBS and used to screen the tissue culture supernatants from the 
various hybridoma clones. Tissue culture supernatants of various dilutions 
from fusion No. 1 or fusion No. 2 were added to microtiter plates in a 
final volume of 50 ul and incubated at 0.degree.14 4.degree. C. for 16 h. 
The plates were then washed and the antibody binding activity was 
determined by alkaline-phosphatase activity conjugated to rabbit antimouse 
antibody. Mouse polyclonal antiserum against polypeptide NMT-1 was used as 
a control (IMS). 
Analysis Of Estrogen Receptor Interaction With Monoclonal Antibodies 
Produced Against Oligopeptide NMT-1 By Sucrose Density Gradients 
Calf uterine cytosol was prepared in buffer TGET/MO and labeled with 10 nM 
.sup.3 H!E.sub.2 at 0.degree. C. for 4 h. To determine non-specific 
binding, aliquots were labeled with .sup.3 H!E.sub.2 in the presence of 5 
uM unlabeled DES. At the end of the incubation, free radioactivity was 
removed with dextran-coated charcoal pellets and samples were removed and 
placed in polypropylene microfuge tubes. Aliquots (50-100 ug equivalent of 
the antibody) of the ascites fluid from the various clones were then added 
and the samples were reincubated at 0.degree. C. for 16 h. Additional 
samples were treated either with an equimixture of ascites from four 
clones or remained untreated (control). Samples were then analyzed on 
5-20% sucrose density gradients made in TGET/MO buffer containing 0.4M KCl 
as described in the methods. 
Results 
Five monoclonal antibodies were developed against peptide NMT-1 (amino 
acids 140-154 of hER). These monoclonal antibodies were found to be 
receptor-specific and exhibited all the characteristics described for the 
polyclonal antisera raised against this peptide. Western blot analysis 
demonstrated that each of the five monoclonal antibodies recognized a 55 
kDa protein extracted from nuclei of estrogen target tissues and from 
human breast tissue samples that were shown to contain cytoplasmic 
estrogen receptor by ligand binding assays. The monoclonal antibodies 
recognize the estrogen receptor in immunocytochemical assay using human 
breast tissue and in rat uterine tissue. The monoclonal antibodies to 
NMT-1 also detect a nuclear protein in the MCF-7 cell line (human breast 
cancer cell line). 
Experimental Series 5 
Interaction Of anti-A/B Domain Monoclonal Antibodies Prepared Against 
Oligopeptide NMT-1 With .sup.3 H!E.sub.2 From Various Mammalian Species 
Several studies have suggested that ER protein has two conserved regions, 
namely the DNA-binding (region "C") and the general steroid binding domain 
(region "E"). Thus, it is possible that the MAbs produced against the A/B 
transactivation domains of hER will also interact with ER from various 
species. To empirically demonstrate such interaction, cytosols from human 
breast cancer tissue, calf uteri and rat uteri were labeled with .sup.3 
H!E.sub.2 for 16 h at 0.degree. C. After removal of free radioactivity 
with DCC, samples were incubated without (control) or with monoclonal 
antibodies NMT-1-C6 and NMT-1-E7. After 16 h at 0.degree. C. the samples 
were analyzed by sucrose density gradients as described earlier. The 
overall results are summarized by Table 3. 
Receptor Specificity Of Anti-A/B Domain Monoclonal Antibodies 
It has been suggested that the DNA binding domain of receptor proteins in 
the steroid hormone family share 42-95% homology with each other Evans, 
R. M., Science 240:889 (1988)!. The production of MAbs to this A and B 
region of human ER thus might result in MAbs which cross-react with 
progesterone receptor, glucocorticoid receptor, and androgen receptor. The 
experiment was conducted to test this possibility. 
Initially, progesterone receptors of calf uterine cytosol were labeled with 
.sup.3 H!ORG2058; glucocorticoid receptors were labeled by incubating 
calf uterine cytosol with .sup.3 H! dexamethasone; and androgen receptors 
of rat ventral prostate cytosol were labeled with .sup.3 H! DMNT as 
follows. 
Cytosol fractions from each tissue were prepared in the appropriate buffer, 
as described previously. Briefly, fresh tissue or frozen tissue powder was 
homogenized (1 g/4 ml) in buffer TT, pH 7.4 at 2.degree. C. The homogenate 
was then centrifuged at 105,000.times.g for 45 min at 2.degree. C. and the 
supernatant fraction (cytosol) was used for receptor binding studies. 
To label the ER aliquots of the calf or human breast tissue, cytosols were 
incubated at 2.degree. C. for 4 h with 5 nM .sup.3 H!E.sub.2 in the 
absence (total binding) or presence (non-specific binding) of a 100 fold 
molar excess of unlabeled DES as previously reported Muller et al. J. 
Bio. Chem. 258:9227-9236 (1983); Muller et al., Cancer Res. 40:2941 
(1980)!. Progesterone receptors were labeled by incubating calf uterine 
cytosol with 15 nM .sup.3 H!ORG2058 in the absence or presence of 
unlabeled ORG2058 as described previously Traish et al. Steroids 47:157 
(1986)!. Androgen receptors were labeled by incubating rat prostate 
cytosol with 10 nM .sup.3 H! DMNT in the absence or presence of unlabeled 
DHT; and glucocorticoid receptors were labeled by incubating calf uterine 
cytosol with 10 nM .sup.3 H! dexamethasone in the absence or presence of 
unlabeled DEX as conventionally known Traish et al. Endocrinology 
118:1327 (1986)!. At the end of the incubation, free radioactivity was 
removed with dextran coated charcoal pellets and the supernatant used for 
antibody-receptor interactions. 
Individual aliquots of these labeled receptor preparations were then 
incubated at 0.degree. C. for 16 h with an aliquot of monoclonal antibody 
NMT-1-C6 and NMT-1-E7. As a control, other aliquoted samples were 
incubated with buffer only. All samples were then analyzed on SDG as 
described previously. The overall results are summarized within Table 3 
also. 
TABLE 3 
__________________________________________________________________________ 
Reaction With Cytosols Of ER From: 
Reaction With Cytosols Of: 
Anti-NMT-1 
Human Breast 
Mouse 
Calf 
Rat Progesterone 
Androgen 
Glucocorticoid 
Monoclonal Antibody 
Cancer Uterine 
Uterine 
Uterine 
Receptor 
Receptor 
Receptor 
__________________________________________________________________________ 
C6 Yes Yes Yes Yes No No No 
E7 Yes Yes Yes Yes No No No 
__________________________________________________________________________ 
A more detailed description of the characteristics of monoclonal antibodies 
NMT-1-C6 and NMT-1-E7 is presented by FIGS. 5-15 respectively. In brief, 
FIG. 5 shows the antibodies from the tissue culture media of these two 
clones. Also these two clones immunoprecipitate ER using the tissue 
culture supernatants. Ascites were produced from these two clones and were 
further characterized. FIG. 6 demonstrates that these antibodies are 
specific for the estrogen receptor. FIG. 7 shows that the antibodies are 
site specific since displacement of the receptor from the antibody was 
made in presence of the peptide. FIG. 9 suggests that these antibodies 
recognize their binding epitope, even the aggregated 8S receptor form. 
FIG. 10 shows that these antibodies recognize the salt-activated 4S ER 
form. Heat transformation which results in 5S homodimerization, does not 
preclude binding of the antibodies; however, heating promotes protease 
activity and as shown in FIG. 11, only a portion of the receptor is bound 
to the antibody. FIG. 12 shows that when transformation of the ER is made 
by ammonium sulfate precipitation, the antibody bound the majority of ER 
dimer. The proteolyzed receptor generated during heat transformation does 
not bind to the antibody suggesting that this epitope is in a domain 
susceptible to degradation (FIG. 13). The binding of the antibody to the 
4S does not interfere with dimerization or transformation (FIG. 14). The 
binding of the antibodies to the A/B region does not interfere with 
binding o the monoclonal antibody 213 to the DNA-binding domain (FIG. 15). 
Also, the binding of these two antibodies together produced a larger 
complex than binding of either alone. 
Experimental Series 5 
Binding Of Monoclonal NMT-1-C6 And NMT-1-E7 To Estrogen Receptor 
Specifically, FIGS. 5A and 5B illustrate ER interaction with antibody from 
hybridoma MAb#NMT-1 C6 and E7. Calf uterine cytosol was prepared in buffer 
TEGT and labeled with 10 nM .sup.3 H!E.sub.2 at 0.degree. C. for 2 hours. 
200 ul of the labeled cytosol were incubated with 200 ul of hybridoma 
supernatants from clone E7 (FIG. 5A), or C6 (FIG. 5B) for 16 hours at 
0.degree. C. 200 ul of buffer was added to the control. Free radioactivity 
was removed by DCC pellet. Samples were then analyzed on 5-20% SDG/0.4M KCl 
made in TEGT buffer. The arrows represent sedimentation of the .sup.14 
C-labeled human serum albumin. Open circles represent the control while 
closed circles represent the addition of the monoclonal antibody. 
Additional data is provided by Tables 4 and 5 respectively. Table 4 
summarizes the immunoprecipitation of .sup.3 H!E.sub.2 -ER complex by 
hybridoma MAb#NMT-1 E7 and C6. Calf uterine cytosol was prepared and 
labeled as described above, but instead of analyzing the incubations on 
SDG, protein G was added and incubated with the mixture for 16 hours with 
agitation of 0.degree. C. As described in material and method, the 
precipitated ER-MAb complex was counted and the percentage precipitated 
was calculated based on the specific binding. Similarly, Table 5 
summarizes the immunoprecipitation of .sup.3 H!E.sub.2 -ER complex by 
MAbs NMT-1 C6 and E7. Samples were prepared the same way as in Table 4 
except the MAb used was from the ascites preparations. 
TABLE 4 
______________________________________ 
MAb .sup.3 H!E2ER % Precipitate 
______________________________________ 
Buffer 7% 
C6 23% 
E7 25% 
______________________________________ 
TABLE 5 
______________________________________ 
MAb % .sup.3 H!E2-ER Precipitate 
______________________________________ 
Buffer 5% 
C6 66% 
E7 63% 
______________________________________ 
FIGS. 6A-6C illustrate the specificity of the MAbs. Calf uterine cytosol 
was incubated either with .sup.3 H!ORG 2058 or .sup.3 H!E.sub.2 for 2 
hours to label progesterone or estrogen receptors respectively. Rate 
prostate homogenized in 0.4M KCl, was incubated (as whole tissue extract) 
with .sup.3 H!DMNT to label the androgen receptor. Aliquots of these 
receptor preparations were then incubated at 0.degree. C. for 16 hours 
with aliquots of the MAb ascites or with buffer as a control, 0.1 volume 
4M KCl was added to all samples, and analyzed on SDG. FIG. 6A represents 
estrogen receptor; FIG. 6B is progesterone receptor; and FIG. 6C shows 
androgen receptor. Open circles represent the control; closed circles the 
MAb; and the arrows represent the bovine serum albumin marker. 
In comparison, FIGS. 7A-7C illustrate the absence of species specificity of 
MAb#NMT-1 as determined by SDG. Cytosols from calf (FIG. 7A) and mouse 
uteri (FIG. 7B) and human breast cancer tissue (FIG. 7C) were prepared and 
labeled as described previously herein. Samples were then incubated without 
monoclonal antibody (open circles) or with monoclonal antibody (closed 
circles). The incubated samples were analyzed on SDG. 
Additional data is provided by Table 6 below. Table 6 summarizes the 
immunoprecipitation of rat-ER by MAb#NMT-1. Rat uterine tissue was 
homogenized in 0.4M KCl; centrifuged; and aliquots from the supernatant 
(whole tissue extract) were incubated in 10 nM .sup.3 H!E.sub.2 for 16 
hours with or without unlabeled DES. Free radioactivity was then removed 
by DCC. Samples were reincubated for 16 hours with MAb#NMT-1, or buffer, 
or progesterone receptor monoclonal antibodies as a negative control. 
Protein G was added next and the mixture incubated with agitation for 
another 16 hours. To subtract the nonspecific precipitation, Protein G was 
also added to the DES treated samples. 
TABLE 6 
______________________________________ 
MAb % Precipitated .sup.3 H!E2 
______________________________________ 
Buffer 1% 
C6 38% 
E7 0.6% 
______________________________________ 
FIGS. 8A and 8B demonstrate the site specificity of MAb#NMT-1 as determined 
by SDG. Calf uterine cytosol was prepared and labeled as described before. 
Aliquots of the anti-A/B domain monoclonal antibody were preincubated at 
0.degree. C. for 2 hours either in the absence (control) shown by FIG. 9A 
or the presence shown by FIG. 9B of 50 mg of NMT-1 peptide in TEGT. 
.sup.3 H!E2 labeled cytosol was incubated for 4 hours at 0.degree. C. 
Each incubation was analyzed on SDG. Closed circles in FIG. 8A represent 
the sedimentation of .sup.3 H!E2 incubated with MAb. Closed circles in 
FIG. 8B represent the sedimentation of .sup.3 H!E2 incubated with MAb 
bound to peptide. Open circles represent the control with no MAb added. 
Experimental Series 6 
Binding Of Monoclonal Antibodies To The Untransformed 8S, Activated 4S, and 
Transformed 5S Isoforms 
FIG. 9 demonstrates the interaction of MAb#NMT-1-C6 with the 
molybdate-stabilized untransformed 8S form of ER. Calf uterine cytosol 
prepared in TEGT buffer containing 10 mM sodium molybdate was incubated at 
0.degree. C. for 2 hours with 10 nM .sup.3 H!E2 in the absence or presence 
of unlabeled DES. Free radioactivity was removed by DCC. Aliquots of the 
cytosol were incubated at 0.degree. C. for 16 hours without (open circles 
as control) or with 10 ml of monoclonal antibody C6. The samples were 
analyzed on SDG in buffer without KCl. The arrow represents ovalbumin 
marker. Experimentals using the E7 monoclonal antibody were substantially 
similar to the data of FIG. 9. The substantial identity of results between 
controls and MAb samples shows that the antibody binding site was 
inaccessible in the native (8S) state of 8 hER. 
In comparison, FIG. 10 illustrates the interaction of MAb#NMT-1-C6 with the 
salt-activated 4S form of ER. Calf uterine cytosol prepared in TEGT was 
incubated for 2 hours with 10 nM .sup.3 H!E2 in the absence or presence 
of DES. After treatment with DCC, samples were incubated for 16 hours at 
0.degree. C. without (open circles as control) or with (closed circles) 
monoclonal antibody C6. All samples were analyzed on SDG. The results of 
FIG. 10 show substantial binding of the monoclonal antibody to the 4S form 
of ER. Experimentals using MAb-E7 were substantially similar to the data of 
FIG. 10. 
Finally, FIG. 11 reveals the interaction of MAb#NMT-1-C6 with the 
heat-transformed 5S form of ER. Calf uterine cytosol prepared and labeled 
the same way as previously except that before treating the samples with 
DCC, they were incubated at 28.degree. C. for 30 min. Aliquots from the 
labeled-transformed ER were incubated with MAb-C6 in the presence of 0.1 
volume of 4M KCl. Open circles represent samples without MAb while closed 
circles represent samples with MAb. Arrows represent the bovine serum 
albumin (BSA) marker. The results show substantial binding of the C6 
monoclonal antibody to the 5S isoform of ER. Experimental using MAb-E7 
shows results substantially similar to the data of FIG. 11. 
Experimental Series 7 
Other Binding Characteristics of Anti-A/B Domain Monoctonal Antibodies 
FIG. 12 shows the interaction of the MAb with the ammonium sulfate 
transformed ER. Aliquots of the ammonium sulfate transformed ER (as 
described in Materials and Methods) were incubated with 10 nM .sup.3 H!E2 
for 2 hours with or without unlabeled DES. After treatment with DCC, 
samples were incubated for 16 hours with 10 ml of the MAbs (closed 
circles) or buffer (open circles) in the presence of 0.1 volume KCl. FIG. 
12 shows substantial binding of the MAb to the ammonium sulfate 
transformed ER. 
In addition, FIGS. 13A and 13B show the results of adding the MAb-C6 and 
the MAb-E7 monoclonal antibodies in the aggregation of the 5S ER in the 
absence of KCl. Calf uterine cytosol was prepared the same way as 
described previously herein, but no KCl was added either to the samples or 
to the SDG. 
Equally important, the data shown by FIGS. 14A and 14B respectively 
demonstrate that MAb#NMT-1 does not interfere with receptor 
transformation. Calf uterine cytosol was prepared and labeled as described 
herein except that the MAb was added either before heat activating the 
receptor (FIG. 14A) or after heating (FIG. 14B). Open circles represent 
that no MAb was added (control) and closed circles show that MAb was 
added. The arrows indicate the sedimentation of BSA marker. 
Lastly, FIG. 15 shows the interaction of both MAb#NMT-1 and MAb#213 (a 
C-domain specific MAb) with salt activated ER. Calf uterine cytosol 
prepared and labeled as described in FIG. 9 was aliquoted into 4 samples 
of 200 ml and incubated for 16 hours at 0.degree. C. with buffer (control 
open circles), or MAb#NMT-1 (closed circles), or MAb#213 (open triangles), 
or a mixture of both MAbs (closed triangles). The samples were analyzed on 
SDG. 
D. The Functional Status Of The A/B Domain Of hER As Determined By Anti-A/B 
Domain Antibodies 
Experimental Series 8 
Detection Of An Altered A/B Region Of ER In Clinical Human Breast Cancer 
Specimens 
An analysis of ER from 29 human breast tumor clinical specimens was 
undertaken with monoclonal antibody #213 (an MAb directed to the ER 
DNA-binding C domain alone) and monoclonal antibody NMT-1C6 (directed 
specifically to the A/B region) demonstrated the presence of altered 
estrogen receptors in these tumors. Based on this analysis, the tumors 
were able to be classified into four groups (I-IV). In group I (10 tumor 
specimens), ER bound both the A/B domain and the C domain specifically 
site-directed antibodies, as demonstrated by density shift of the 
ER-antibody complexes shown by FIG. 16A. In group II (3 tumor specimens), 
while the ER DNA-binding domain remained intact, the A/B region subdomain 
was functionally altered, as shown by the density shift of ER with 
monoclonal antibody #213 (the C domain MAb), but not with the NMT-1C6 
specific for the A/B domain, as illustrated by FIG. 16B. In group III (13 
tumor specimens), however, the DNA-binding C domain appeared defective, 
while the A/B region subdomain remained intact, as determined by density 
shift of ER with NMT-1C6 antibody, but not with monoclonal antibody #213 
--as shown by FIG. 16C. In group IV (3 tumor specimens), ER did not bind 
either of the two antibodies, showing that ER in these tumors is altered 
in both the DNA and the A/B subdomains. This last result is shown by FIG. 
16D. 
E. Conclusions And Summary 
The experiments and empirical data presented herein unequivocally show that 
the functional status of the A/B region and the activation and 
transformation capability for the hER protein can be determined using the 
structural isoform differences existing among the native (8S), activated 
(4S) and transformed (5S) isoforms of hER. While the polyclonal antisera 
do not show any differences in binding specificity and are thus unable to 
discriminate structurally among the three isoforms, the anti-A/B region 
hER monoclonal antibodies do provide such a discriminatory capability. The 
relationship between structural state and functional status is based--not 
on the presence of absence of particular amino acid residues in 
sequence--but rather on the distinctive ability to identify and 
distinguish among the 8S, 4S, and 5S isoforms of hER having the same amino 
acids in sequence. Clearly, the monoclonal antibodies provide a proven 
capability to bind specifically with a single epitope within the A/B 
domain in the activated but untransformed (4S) form and in the activated 
and transformed (5S) form of hER; however, they do not bind with the 
native (8S) form of hER. Thus, the basis for evaluating and determining 
the functional status of the A/B domain of an hER sample under test, lies 
in the power to identify and discriminate among the three isoforms of hER.