Note: Descriptions are shown in the official language in which they were submitted.
1(~811Zl
8ACKGROUND O~ THE I NVENT I ON
_ The present invention relates to a method and
means for the determination of the hormones human chorionic
gonadotropin (HCG), luteinizing hormone (LH) and prolactin
(PRL) and more especially to a fundamentally new method and
means for detection of pregnancy in the human female.
Tests for the detection of pregnancy are generally
based on the determination of hormones which are produced by
the developing placenta, such as gonadotropic hormones simi-
- 10 lar to those produced by the anterior pituitary gland and
steroid hormones similar to those of the ovary and adrenal
- gland. Pregnancy tests in use today are nearly exclusively
based upon an assay for the placental hormone, human chorio-
nic gonadotropin (HCG). This hormone is found in body fluids
(blood serum and urine) only during pregnancy, with the
exception of several other very rare hormone-producing
conditions of the body. The International Unit (I.U.) of
HCG was adopted in 19~8 and is defined as the specific
gonadotropic activity of 0.1 mg. of a dried standard kept at
the National Institutes of Health, London, England.
The earliest tests for pregnancy were based upon
biological In vivo methods for determining the presence of
HCG. For example, the earliest test, the Aschheim-Zondek
test was based upon the ability of HCG injected subcutaneously
in mice to produce corpora lutea. The Friedman test is
another biological test in which a urine sample of the sus-
pected pregnancy is injected into the ear vein of a mature
femal rabbit which has been isolated 3 to 4 weeks, and 48
hours after injection the ovaries are examined for ruptured
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hemorrhagicfollicles, which ir-ldicate a positive reaction.
A lesser known test developed by Kupperman in 191l3 involves
the injection of the patient's urine into a female rat with
subsequent inspection of the ovary for signs of hyperemia.
While the two hours necessary for conducting this test is
considerably shorter thant the 48 hours required for the
Friedman test and the nearly five days required for the
Aschheim-70ndek test, this test is not as reliable since
it requires a skilled technician to differentiate a slightly
pink negative ovary from a reddened positive ovary to attain
a high degree of accuracy. A test developed by Galli and
Mainini in the late 1940's also requires only approximately
two hours to conduct the test; however, this test involves
the injection of the patient's urine into frogs with the sub-
-~ 15 sequent observation for ejection of sperms, and these animals
are relatively insensitive compared with rabbits, mice and
rats.
All of the foregoing biological tests suffer from
serious disadvantages, including the availability of animals,
the need to maintain a large colony of animals, and relatively
long periods of testing, frequent yielding of false positive
and negative results, and most significantly, the fact that
a positive test can be achieved with only a 95~ degree of
reliability only after a period of 25 to 30 days following
ovulation.
A second generation of pregnancy tests developed
during the early 1960's are characterized as immunological
or immunochemical procedures. Since HCG is a protein horn-one,
it acts antigenically in a heterologous species. Accordingly,
1081121
when HCG is injected by suitable techniques into an appro-
priate test animal, most typically a rabbit, an antibody
to HCG is produced within the animal. In early tests
utilizing this principle, it was attempted to utilize the
antigen-antibody direct precipitin reaction, according to
which a visible precipitate would form as a result of the
combination of HCG and its antibody, to detect the presence
of this hormone. More common procedures involved so-called
indirect methods of determination, such as the latex particle
slide test of Brody and Carlstrom and hemagglutination and
inhibition test of Wide and Gemze1. In the former procedure,
an antiserum is added to the urine of a patient followed by
a latex carrier which has been coated with HCG. If the
urine specimen is of a pregnant woman and contains HCG, the
antibodies in the antiserum will be neutralized and will not
therefore react with the HCG coated on the carrier to pro-
- duce agglutination. In the latter testing method, a similar
principle is employed except that the indicator comprises
HCG conjugated with red blood cells or formalinized red ~ -
blood cells. The first test may be carried out in a period
of only about two minutes whereas the second requires appro-
- ximately two hours. Either the patient's urine or blood
may be employed for these test procedures.
In a very recently developed pregnancy testing
procedure based upon a modification of the basic immunolo-
gical mechanism, radiological means are employed to detect and/
or measure the presence of HCG in the patient's blood (or
urine). See, for example, Goldstein et al. in Fert. Steril.,
Vol. 23, page ~17 (19'72). In these radioimmunoassay tests,
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the antibody is placed in contacl with a mixture of the
patient's body fluid to be tested and a known amount of ~-
HCG tagged with a radioactive isotope, and the HCG in the
test sample and the labeled HCG compete for interaction
with the HCG antibody. The antibody is then separated
from the fluid and either fraction may be analyzed radio-
logically to determine the respective proportions of the
labeled and unlabeled HCG which became bound to the anti-
bodies, and the concentration of HCG in the sample can be
calculated from this information since the proportion of
labeled and unlabeled HCG will be in the same proportion in
both fractions. The radioimmunoassay techniques have over-
come one significant limitation of the immunochemical preg-
nancy test, namely, that of sensitivity. Radioimmunoassay
techniques are several thousand times more sensitive than
the above-described indirect tests, and accordingly, permit
a detection of pregnancy much earlier than the 25 to ~0 days
following ovulation required with the latex particle slide
- test and the hemagglutination test. However, even though
pregnancy may be detected after the 10th or 12th day follow-
ing ovulation with a 95~ degree of reliability by the radio-
immunoassay method, these tests have the disadvantage that
they require more time to carry out, typically about 24-48
hours.
The most serious drawback, however, with all pre-
viously known pregancy testing techniques involves the
frequent indication of false positive and negative results.
In the case of the immunochemical pregancy test, this
difficulty is due to non-specific immune response related to
non-specific antibody-antigen reactions. For example, the
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presence of a common hormone, non-specific alpha-subunit
amoung fo11icle stimulating hormone (FSH), luteinizing
hormone (LH), HCG and thyroid stimulating hormone ~TSH),
and homologies in the amino acid sequence of the hormone-
specific beta-subunits have caused further difficulties in
producing specific antisera for use in the immunochemical
techniques. These difficulties are exaggerated in radio-
immunoassay methods due to the high degree of sensitivity
of this technique. This last-mentioned drawback has been
partially circumvented by prodcing antisera specific to
the beta-subunit of HCG; however, this manipulation requires
the use of valuable material, immunization, selection and
purification to achieve specific antibodies, which procedures
are time consuming, expensive and very cumbersome. In spite
of these precautions, the goal of near 100~ reliability in
the detection of pregnancy by radioimmunoassay has not been
accomplished.
Furthermore, a serious need exists for a means to
` detect ectopic pregnancies at the earliest possible moment.
Such pregnancies have negative hemagglutination or latex
slide tests in 40 to 60~ of all patients, even after the 25-30
day period following ovulation required to perform these tests.
The reason for this relates to the belief that the immuno-
chemical pregnancy testing techniques provide for detection
of a pregnancy only after implantation of the fertilized -
ovum has occurred. As described hereinabove, all of the
heretofore known pregnancy testing methods suffer from this
disadvantage that they are not effective until after nidation.
A test which could determine the presence of a pregnancy
during the period between fertilization of the ovum and ~ "
5 - -
1~)81~
implantation of the fertili~ed ovum would be invaluable in
the treatment of, for example, threatened abortion in women
who habitually abort, cases involving artificial insemina-
tion and in connection with new contraceptive methods which
effectively terminate a pregnancy prior to implantation.
The presence of specific receptors for the
various hormones has long been suspected in target cases of
both humans and animals, and reseachers have had success
within the last half decade in identifying and directly
studying certain of these receptors and their interaction
with many respective hormones. Preliminary studies under-
taken by the present inventor have suggested the presence
of a receptor for HCG in the corpus luteum of pregnant or
pseudo-pregnant rats. Gonadotropins, Chapter 21, 1972,
John Wiley and Sons. These preliminary studies, however,
gave no indication of whether a similar receptor is present
in humans or other animals, whether the receptor found in
rats is species specific or specific even for HCG, whether
it would be a reliable indicator for HCG in body fluid
samples or whether it is stable over any extended period
of time.
Prolactin (PRL) represents a hormone which for
many years was not believed to be present in humans. Its
presence in humans was confirmed in the early 1970's. The
presence of PRL receptors in mammary tissue has been documented
and the hormone is believed to play a role in breast cancer,
pituitary tumors, lactation disorders, hypothyroidism and
other disorders. There are thought to be as many as o~
different functions of PRL involving complex interaction
with other pituitary gonadal and adrenal hormones, possibly
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~08112~
in the role as an intermediary regulator for thes~ other
hormones. A luteotropic as well as a luteolytic role of
PRL during the estues cycle of the rat has also been sug-
gested, and there is some evidence developed in the last
few years regarding studies of the rat. The specific bind-
ing of PRL to rat ovary has been suggested very recently in
the literature, but no specific receptor has been identified.
Turkington et al., Rec. Prog. Horm. Res., 29:417 (1973).
Studies by several investigators have suggested that PRL
has little function in the maintenance of corpus luteum in
humans. Hwang et al., Proc. Nat. Acad. Sci., 60:1902 (1971);
Midgley et al., Proc. IV Int. Cong. Endocrinal, Int. Cong.
Series No. 273, Excerpta Medica, Amsterdam, 1972.
In view of the maladies identified above which
are known to involve the hormone PRL, it is desired often-
times to determine and/or monitor the PRL content in a test
sample obtained from a subject, for example, a sample of
body fluid. In some cases, only small samples are avail-
- able, e.g., pediatric cases, and therefore, it is desired
to have a very specific test which requires only a small
sample and only a short period of time to carry out.
Specificity is desired in order to differentiate from other
hormone species which may be present in the sample, and
this is particularly true in cases where it might be desired -
to simutaneously determine or measure two or more different
hormones. There exists at present no truly specific means
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for making such a determination.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention
- to provide a method for the determination of human chorionic
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gonadotropin (HCG), luteiniziny hormone (LH), prolactin
(PRL) and HCG-like materials in an aqueous sample.
Another object of the present invention resides in
the provision of a pregnancy testing method which produces
nearly 100 reliability in detecting pregnancy.
It is also an object of the present invention to
provide a method for the detection of ectopic pregnancy and
'threatened abortion wherein the pregnant condition may be
ascertained 6 to 8 days following inception.
A further object of the present invention resides
in providing a pregnancy detection method according to which
a pregnancy can be detected as early as the sixth to eighth
; day following ovulation with almost one hundred percent
reliability.
It is additionally an object of the present invention
to provide a means for determi,ning the presence of the hor-
mone HCG in an aqueous sample, particularly for the purpose
of recognizing pregnancy in the human female by investigat-
ing a sample of body fluid.
' ? Another object of the invention resides in the
provision of a method for measuring the content of PRL in an
- aqueous sample, especially in a sample of body fluid.
It is also an object of the invention to provide
a method and means for simultaneously determining HCG, or LH
and PRL in an aqueous sample, particularly a very small sample
of body fluid.
A further object of the present invention resides
in providing the particular receptor employed in the radio-
receptorassay method set forth above.
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It i5 also an object of the present invention to
provide a method for producing the specific receptor em-
- ployed in the radioreceptorassay method referred to above.
In accomplishing the foregoing objects, there
has been provided in accordance with the present invention
a method for the determination of human chorionic gonado-
tropin (HCG), luteinizing hormone (LH) and HCG-like material
and/or prolactin (PRL) in an aqueous sample involving the
steps of contacting such a sample with an agent capable of
selectively binding the hormone, providing a means of in-
dicating whether such binding has taken p1ace and observing
the indicating means to determine the presence of the
- hormone in the sample, wherein a particularly improved
aspect comprises the binding agent being a plasma membrane
extract from the corpus luteum of an animal which possesses
the receptor for the hormone HCG and/or PRL. The indicator
system employed in this method may be any of those conven-
; tionally employed in a similar immunochemical method for
determining proteins and/or polypeptides. For example, the
indicator may be a material which has been treated with the
hormone so that in the presence of the instant receptor,
the indicator will discolor, become colored, agglutinate,
precipitate or give some other visible indicat;on (for
example, fluoresence) of the presence or absence of the
hormone in the fluid being tested, or preferably, the more
accurate radiological measuring techniques may be employed.
- In applying this method for the detection of pregnancy in
human females, a blood or urine sample is contacted with
the above-described specific receptor and the presence or
absence of HCG in the sample is determined based upon the
amount of binding evidences by the indicator system.
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In a preferred embodiment of the present invention,
there is provided a method for the determination of HCG in
an aqueous sample~ preferably the blood serum or urine of a
patient having a suspected pregnancy, comprising contacting
a highly specific receptor for the hormone HCG, which recep-
tor is a plasma membrane extract from the corpus luteum of
an animal possessing the receptor for this hormone, with the
aqueous sample having added thereto an amount of HCG which
has been labeled with a radioactive isotope. In this method,
the possible HCG present in the sample and part of the radio-
actively labeled HCG are bound to the receptor. The receptor
with its bound HCG is then separated from the aqueous sample
and the radioactivity of one or both of the separated frac-
; ,
tions is measured to determine the concentration of HCG in
the aqueous sample, this concentration being a function of
the measured radioactivity. It is preferred to obtain the
receptor from the corpus luteum of a cow, sheep,horse or
hog, particularly from an animal which is pregnant, and
most preferably an animal during the first trimester of
pregnancy.
A further embodiment comprises a method for deter-
mining the hormone prolactin (PRL) in an aqueous sample,
again preferably a body fluid, including the steps of con-
tacting the sample with a highly specific receptor for PRL,
this receptor also being a plasma membrane extract of the
corpus luteum of an animal possessing this receptor, and
providing in the sample an amount of PRL labeled with a
radioactive isotope. The remaining steps are identical
to those set forth above for determining HCG. This method
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can be used as an indication of pregnancy in a human remale
or for indicating other disorders involving the presence
of prolactin.
There is also provided in accordance with the
invention a method for simultaneously determining the two
or more different hormones contained in a single aqueous
sample. In this embodiment, the steps are similar to those
for measuring single hormones, except that the aqueous sample
is contacted with one or more agents capable of selectively
binding each of the hormones to be determined, providing a
separate and distinguishable means for indicating whether
binding has taken plac-e for each hormone and observing each
of the indicators to determine the presence of each hormone.
The binding agent is a plasma membrane extract from a body
organ of a species which is known to possess in the organ ~ -
a specific receptor for each of the hormones to be measured.
Preferably ~he receptor is a plasma membrane extract of the
corpus luteum of a mammal, which possesses receptor sites
for both HCG and PRL. The preferred indicating means con-prise
different radioactive isotopes, for example, different iso-
topes of iodine such as l251, l311, 3H and l~C. With such an
indicator, HCG and PRL can be simulataneously measured by
contacting the receptor with the sample and differently
labeled HCG and PRL, so that part of the labeled and un-
labeled hormones are bound to the receptor, separating the
bound and unbound hormone from the sample and counting the
radioactivity of each isotope species to determine the
presence of the respective hormones in proportion to the
radioactivity measured. ~-
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- In another en-bodiment of the present invention,
there is provided a reagent for receptor-chemical deter-
mination of the hormone HCG, which reagent comprises in
substantially pure form the specific fraction of plasma
membrane extract from the corpus luteum of an animal pos-
sessing the receptor for HCG, the specific fraction being
one which is capable of selectively binding biologically
active human chorionic gonadotropin.
In yet another embodiment of the present inven-
tion, there is provided a method for the preparation of a
reagent for receptor-chemica1 determination of the hormone
HCG, comprising the steps of preparing a finely communited
tissue homogenate from corpus luteum tissue of an animal
which possesses the receptor for HCG, separating the plasma
membranes from said homogenate and selecting the plasma
membrane fraction capable of selectively binding biologi-
cally active human chorionic gonadotropin.
The subject testing procedure is also suitable
for detecting pregnancy in animals. Accordingly, it may
be used to detect pregnancy in any of the animal species
wherein the specific receptor of the invention is found,
i.e., mammals and possibly selected non-mammals.
A further embodiment of the invention comprises
means for the determination of human chorionic gonadotropin
(HCG), luteinizing hormone (LH) or HCG-like material in an
aqueous sample, comprising (a) a first reagent and a second
reagent; (b) said first reagent comprising in substantially
pure form the specific fraction oF plasma membrane extract
from the corpus luteum of a species having the receptor for
human chorionic gonadotropin capable of selectively binding
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. biologically active human chorionic gonadotropin; (c) said
second reagent comprising labeled human chorionic gonado-
tropin capable of emitting radiation, said first reagent
I bçing intended to be contacted with the sample containing
¦ 5 the hormone to be measured and with the second reagent to
bind part of the labeled and unlabeled hormone to said
receptor; (d) means for measuring the amount of labeled
hormone bound to said receptor, the emitted radiation there-
~ro~ irl~J .1 lullr~ir~ r~l' ll~r~ (~ol)r~ oll ol ll~ llllr~
in the aqueous sample. This embodiment preferably takes
the form of a pregnancy testing kit.
Also provided according to the invention is a
means for the determination of prolactin (PRL) in an aqueous
sample, comprising (a) a first reagent and a second reagent;
(b) said first reagent comprising in substantially pure form
the specific fraction of plasma membrane extract from the
corpus luteum of a species having the receptor for prolactin
capable of selectively binding biologically active prolactin;
(c) said second reagent comprising labeled prolectin capable -
of emitting radiation, said first reagent being intended to
be contacted with the sample containing the hormone to be -I
measured and with the second reagent to bind part of the
labeled and unlabeled hormone to said receptor; (d) means
for measuring the amount of labeled hormone bound to said
receptor, the emitted radiation therefrom being a function
of the concentration of the hormone in the aqueous sample.
Finally, in another embodiment of the invention
there is provided a means for the determination of a plurality ~ -
of hormones in an aqueous sample, comprising (a) a first
reagent and a number of second reagents equal to the number
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of hormones to be determined; (b) said first reagent com-
prising in substantially pure form the specific fraction
of plasma membrane extract from a body organ of a species
having the specific receptor in said organ for selectively
binding each of said hormones in biologically active form:
(c) said second reagents comprising each of said hormones
- to be determined in a labeled form capable of emitting
radiation, said emitted radiation being different for each
labeled hormone, said first reagent being intended to be
contacted with the sample containing the hormone to be
measured and with the second reagents to bind part of the
labeled and unlabeled hormones to said receptor; (d) means
for measuring the amount of labeled hormones bound to said
- receptor, the emitted radiation f~ m each 1abeled hormonebeing a function of the concentration of the respective
hormone in the aqueous sample. In this embodiment, the
first reagent is preferably the plasma membrane extract of
a mammal and the second reagents comprise HCG and PRL
labeled with different isotopes.
Further objects, features and advantages of the
present invention will become apparent from the detailed
description of the invention which follows, when considered
with the attached drawing.
BRIEF DESCRIPTIûN OF THE DRAWING
; 25 Fig. 1 is a logit-log linerarization of the
competitive inhibition response of the readioreceptorassay
of HCG, serving as a calculation standard for the method of
the present invention;
14
1083~1Zl
Fig. 2 illustrates the computer output of loyit-
log transformation of the standard for the readioreceptor-
assay of HCG; and
Fig. 3 is a comparison of the plasma levels of
HCG during two normal (HD, RS) and one induced normal intra-
uterine pregnancy in S.L. with HCG levels in 10 patients
with ectopic pregnancy.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there
has been discovered a novel and very specific method for
determination of the hormone human chorionic gonadotropin
(HCG) and/or prolactin (PRL). In contradistinction to
previous biological tests for HCG which have proved to be very
impractical and to more recently developed immunochemical
tests which are based upon the principle of antigen-antibody
binding but which are not sufficiently reliable, the pre-
sent method is based upon the use of a highly selective
receptor for HCG. The receptor is isolated from the target
; tissue of an appropriate animal, whereas in the case of
immunochemical methods, an antiserum is produced from the
blood of a suitable animal which has been injected with
- the hormone to produce antigen-antibody response.
Prior to the present invention there has been
- no acknowledged reason for determining PRL in humans, and
hence, no emphasis on devising suitable-procedures. The
invention is based upon discovery of a specific receptor
for this hormone. The use of a specific receptor isolated
from the target tissue of an animal which possesses this
specific receptor overcomes, apparently entirely, the draw-
back of non-specific responses which often occur in conjunc-
tion with immunochemical procedures. In most cases the
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specific receptor binding recluires the natural configura-
tion of a hormone in its biologically active form. Accord-
ingly, when the receptor-chemical testing procedure is
combined with radio-assay indicating techniques, there
results a radioreceptorassay method which possesses the
degree of sensitivity of the radioimmunoassay techniques
and at the same time the degree of specificity of bioassay,
and therefore, this method evidences greater sustained
accuracy over all existing immunological and biological
hormone tests, particularly tests for pregnancy.
Furthermore, because the receptor requires the
biologically active form of the hormone for binding, the
endogenous synthesis of a metabolically defective HCG,
which normally would give a positive reaction in accordance
with the immunochemical testing procedures but which is
assumed to be incapable of sustaining pregnancy, would
not give rise to a false positive test in accordance with
the testing method of the present invention.
;~ As pointed out in the introduction, the hormone
prolactin has been suggested as having some function in
the estrus cycle of the rat, but at the same time has not
been identified as having any role in the ovarian or
reproductive processes of humans. As a basis for this
invention, it has been discovered that there is an increase -
in the level of PRL in the female during pregnancy. Further- -
more, it has been discovered that ovarian tissue from several
species, including the cow and humans, contains specific
receptor sites for the hormone PRL, in addition to receptor
sites for HCG.
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l Thus, in addition to L~rovidiny a gencrally
applicable procedure for determining PRL in connection with
the various maladies mentioned above, it is also possible
to employ the method oF the invention for determining, or
preferably aidil1g in the determination of pregnancy. A
detected increase in the PRL level in a woman suspected of
being pregnant gives a further indication, together with
the determination of HCG in accordance with the invention,
- that pregnancy in fact exists. This added deyree of con-
firmation is a great value, particularly, in cases involving
abnormal preynancy or threatened abortion. It is bclieved
that PRL plays an important role in fetal development, and
accordingly, its determination provides further information
to that obtained by a determination of ~ICG.
In a preferred aspect of the present invention, it
is possible to simultaneously determine both 11CG and PRL,
since it has been found that the same receptor possesses
- receptor site-1 for both of these hormones. This is a
~ particularly unexpected discovery in view of the fact that
-~ 20 receptor sites for PRL have not heretofore been confirmed
in the corpus luteum of any animal and receptor sites for
-:
HCG have been shown only in the rat. Simultaneous detection
has obvious advantages in pregnancy testing in view of the
¦ above-in~icated role of PRL in pregnancy. A single body
¦ 25 fluid sample and a single testiny procedure provide the
most complete and most accurate indication of pregnancy
available.
This simultaneous determination technique is not
limited to the hormones ~ICG and PRL. For example, it is
known that mammary tissue contains receptor sites for more
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than one hormone, e.g., estrogen, prolactin and oxidocin,
and therefore the technique of the invention is equally
applicable to simultaneous determinations of these hormones
using a receptor derived from breast tissue.
It has also been discovered in accordance with
the present invention that the radioreceptorassay technique
enables the detection of pregnancy as early as one week
following ovulation. This represents indeed a significant
¦ advance in the field of pregnancy determination tests,
¦ 10 since heretofore the pregnancy condition could not be
detected earlier than about the tenth or twelfth day follow-
ing ovulation. This result is more significant when it is
realized that the vast majority of pregnancy tests are
carried out today not by means of the radioimmunoassay pro-
cedure which permits determination as early as ten or
twelve days following ovulation, but rather by means of the
hemagglutination or latex slide tests, which as pointed out
hereinabove permit a determination of pregnancy only after
about 25 or ~0 days following ovulation. Radioimmunoassay
tests require at least 24 hours of laboratory time and
typically about 48 hours of testing time, coupled with
cumbersome and expensive procedures for preparing sufficiently
specific antibody or antiserum. On the other hand, the time
required to carry out the testing procedure in accordance
.
with the present invention amounts to no more than about
one hour and typically only about one-half hour. The
practical advantages flowing from such a pregnancy testing
method which permits determination of the pregnancy condi-
tion prior to implantation of the fertilized ovum in the
endometrial tissue have been briefly discussed above, and
these advantages as well as the significant benefits in
terms of basic research in the field of human reproduction
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resulting from the present invention are readily apparent
to those knowledgable in this art.
In view oF the fundamental similarities in the
binding mechanism of the instant receptor-chemical testing
mechanism and the well-known immunochemical testing mechanism
it is possible to utilize the previously developed indica-
tion mechanisms within the context of the present invention.
Accordingly, the indicator system may consist of an indica-
tor material which has been treated with HCG so that in the
presence of the receptor, the indicator material will dis-
color, become colored, agglutinate, precipitate or give
some other visible or chemical indication of the presence
or absence of HCG in the aqueous sample or body fluid being
tested. From the foregoing, however, it is obvious that
the most effective results are obtained when a radiologocal
assay technique is employed as the indicator system.
Accordingly, the radioreceptorassay embodiment oF the
`~ present invention represents clearly the most preferred
emobodiment. Nonetheless, it is to be understood that the
present invention does not reside in the provision of a
particular indication system for use in the subject method,
but rather in the fundamental and novel employment of
receptor-chemical techniques and a specific receptor to
determine the presence of HCG and/or PRL.
In view of the extreme degree of specificity
; and sensitivity of the radioreceptorassay method of the
present invention, it is possible to make a satisfactory
determination on an extremely small sample. For example,
in utilizing the method of the present invention as a
test for pregnancy, it is possible to perform the test
utilizing only about 0.1 ml. or less of blood taken from
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the patient. This withdrawal from the patient can be
accomplished by means of a simple pinprick in the end of
a finger. Furthermore, the method of the invention is
effective to determine the presence of HCG in any aqueous
sample, and accordingly, either blood serum or a urine
sample from a patient may conveniently be employed.
The specific receptor employed in the testing
procedure according to the present invention is derived
from ovarian tissue. As pointed out hereinabove, the
receptor is significantly more selective for HCG and HCG-
like materials or PRL than is an HCG or PRL antibody,
since the receptor requires the vative configuration of the
hormone in its biologically active form for binding. Hence,
there is no possibility of non-specific immune response.
In all animals which possess this specific receptor, it is
readily detectable in the ovarian tissue which represents
the target tissue of the hormone. While the receptor may
be isolated from any mammal and possibly from certain non-
mammals, its is preferred to obtain the receptor from the
ovaries of relatively large animals, such as cows, sheep,
pigs, horses and the like in view of the fact that these
animals have proportionately a greater amount of ovarian
tissue, and furthermore, a ready supply of such tissue ~
exists by virtue of the fact that these animals are commer- ~ -
cially slaughtered for their meat. It has been determined
that up to 200 pregnancy tests can be performed from a
single cow ovary. The ovaries, for example, of rats are
so small as to effectively render impractical the use of
animals of this type as a source of receptor. As discussed
below, the testing method of the invention is also applicable
. .
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10811:~
to animals which possess the receptor, and there~ore,
positive evaluation of pregnancy in a given species also
provides a method for screening animals for the presence
of the receptor.
Preferably, the animal from which the receptor is
obtained is in a pregnant condition at the time of slaughter,
since at this time the amount of ovarian tissue and the
number of receptor sites is at a maximum. The first trimester
of pregnancy has been found to be the most advantageous
time for obtaining the tissue.
; The specific receptor of the invention is obtained
by separation of pure plasma membranes from the ovarian
tissue followed by evaluation of the various plasma membrane
fractions by testing against isotopically labeled HCG or PRL
and selecting the fraction which shows the greatest binding
for the hormone. The receptor is extremely stable and can
be stored for long period of time, or may even be reused in
a second or subsequent testing procedure. The plasma mem-
brane receptor is stable after lyophilization for pregnancy
test. The exact procedure for obtaining the receptor is
described mor fully below.
Since the testing procedure of the invention is
not species specific, it may also be utilized to detect
pregnancy in any of the animals which have been found to
possess the specific receptor of the invention. Thus, even
though the receptor is very specific for HCG in humans and
LH in humans and animals, it is also capable of selectively
binding HCG-like proteins which play in animals a role
corresponding to HCG in humans, i.e., they correspond to
the chorionic gonadotropins in humans. The same holds
- 21 -
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1()8112~
true for PRL. The advantage to this is that standard
laboratory test animals, such as monkeys, dogs, rabbits,
mice, etc., can be tested by this method whereas separate
antibodies were required for each animal in accordance
with immunochemical testing procedures.
Labeling of the HCG or PRL with a radioactive
isotope can be effected in the conventional manner, with
a suitable isotope for this purpose being selected, for
e ample from 1l3l C1~, H3 and the like. A particularly
suitable isotope is a radioactive isotope of iodine such
as; 125 in view of the fact that labeling with this isotope
is relatively simple and many laboratories possess the
equipment necessary to measure this isotope.
In the case of simultaneous hormone determination
by the receptor assay technique of the invention, it is
necessary to label each hormone to be determined with a
distinguishable isotope, in order that measurement of the
bound and/or unbound hormone fractions will provide a
separate indication for each hormone to be determined. For
example, when conducting a simultaneous determination of
HCG and PRL, purified samples of these two hormones can
be labeled with 125; and l3l1, respectively, and readily
available counting devices can then be used to separately
count each isotope.
In order to more completely describe the present
invention, there are presented below several specific
methods for preparing the test reagents and application of
the radioreceptorassay as well as specific examples of
clinical determinations utilizing the present invention,
it being understood that the specific procedures are in-
tended merely to be illustrative and in no sense limitative.
lO~llZ~ `
l 1. Radioisotopic Labcling of llormone
Highly purificd ~3CG (containing 12,0()0 I.U./mg.
was utilized for the testing. The HCG was labeled with
l by using lactoperoxidase prepared from milk (RZ = 0.78;
Sigma Company, St. Louis, Missouri). Two mci of l in
20 ul. of 0.1 M. sodium acetate buffer of pll. 6.0 were
mixed with 25 ug. of HCG in a reaction vial. An aliquot
of 50 ng. lactoperoxidase in 20 ul. and 200 ng. of ~1202
in lO ul. of water are then added -to the vial. Three
aliquots of l(!0 ng. of II202 were added at five minute
in-tervals. T~e end o~ 20 minutes, the reaction was stopped
by the additionof,0.5l~.of 0.15 M. NaCl containin~ 1%
of bovine serum albumin (~SA) of pH. 7Ø The labeled
hormone was,then separated from free iodine by gel filtration
on a lx30 cm. column of *Sephadex G-100 equilibriated
' with 0.15 M. 2~aCl containing 1% of BSA, pl~. 7Ø The
.
specific activity of ~he labeled IICG was determined by
precipitation with trichloroacetic acid. Five ul. of the
crude reaction mixture was diluted to 5 ml. with 0.05 2~.
~, 20 phosphate buffer of pll. 7.5 containing 0.1% BSA. To a 200
ul. aliquot o~ this solution were added 400 ul. of tri-
chloroacetic acid to a final concentration of 10~ tri-
chloroacetic acid. The precipitated proteins were recovered
by centrifugation. The radioactivity associated with the
precipitable material was considered as protein bound and
was used in the calculation of the specific activity of the
labeled hormone. The crude reaction mixture was purified
by gel filtration through a lX30 cm. column of *Sephadex
G-50 equilibrated with 1% BSA in 9.0 NaCl. The specific
' 30 activity of the I~CG used in the radioreceptorassay was
*Trade Mark ' - 23 -
,. : - , , , , . :
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1~)811Z3L
1 20-30 uCi/ug. The bioloyical activity of the labele(l I{C~
used in the test, as determined by ovarian ascorbic acid
depletion ass~y, was 8,923 I.U./mg. with 95~ confi~ellce
limlts of 5,826-12,250 I.U./mg.
HPRL was iodinated by the method of Hunter and
Greenwood with minor modifications: To 50 ul of 0.5 m
phosphate buffer, p~i 7.5 were added 1 mCi of Na 1 (New
England Nuclear, Boston, ~1assachuset-ts), 5 ug hPRL, and 70
ug of chloram;ne-T, followed after 15 second by sodium
metablsulfite. One hundred mg of iobeads (llycel Reagents,
~louston, Texas) were added to the crude iodinated mixture
: to adsorb unreacted 1. hPRL was also iodinated enzy~ati-
cally by lactoperoxidase (Sigma Chemical Company, St. Louis,
Missouri). Purification was achieved by gel filtration on
a *Sephadex G-100 co]umn (0.7 x 18 cm). The specific activity
and purity of the labeled PRL were determined by chromato-
electrophoresis. l-hPRL capable of binding specifically
to mammary tissue from lactating rat was used for the bind-
ing studies w;th partially purified ovarian homogenate.
II. Preparation of Receptor
- All products in the preparation of plasma membranes
were carried out in an ice bath or at 4C. Fresh bovine ~ -
ovaries from early pregnancy (first trimester: fetus length ~
from crown to rump up to 22 cm.) were obtained from the - ~-
slaugher house and stored in liquid nitrogen until processed.
In a typical experiment, 100 g. of tissue from appro~imately
25 large corpora lutea were cut illtO small pieces with a sharp
blade and ground in 500 ml. of 10 m~5. Tris-liCl buffer of pH.
7.8 containing one mr~. of MgC12, one M~l. dithiothreitol, 10,000
I.U. of *Trasylol (FBA Pharmaceuticals) perliter and 0.25 M.
sucrose. The homogenate was filtered through two layers of
*Trade Mark - 24 -
1081~
1 cheese cloth and the larger particles were reprocessed in 500
ml. of the buffer. The tissue was further homoginized by 10 to
15 strokes in a *Teflonglass homogenizer, Type C with clearance
size of 0.12 to 0.17 mm. at 4C. The homogenate was centrifuged
at 650 xg. for 20 minutes in a *Sorvall refrigerated centrifuge
(RC B, Rotor GSA) to remove intact cells, cell debris and nuclei.
The 650 x g. supernate was again centrifuged at 13,000 g.
for 20 minutes in the same centrifuge. Supernate was this time
discarded and the pellet was resuspended in 50 to 70 ml. of
the Tris-HCl buffer with the aid of the *Teflon-glass
homogenizer. This suspension was injected in the core of
a Tl-14 zonal rotor (Model Beckman Spinco L3-50) spinning at
3,000 rpm. and containing 500 ml. of a linear sucrose gradient
(LKB Ultrograd 11300 pump) from 30% (W/v) to 50% (W/v) sucrose
and 120 ml. of cushion of 50% (w/v) sucrose (pumping rate 20 ml.
min.). An overlay of 20 ml. was injected after the sample.
After two hours of centrifugation at 45,000 rpm., the centrifuge
was decelerated and the rotor content displaced with 55% sucrose
at 20 ml./min. The plasma membranes were displaced from the rotor
in the order of increasing particle size, and fractions of 12 ml.
were collected and immediately frozen until assayed for
binding and enzomatic activity. Membranes were kept frozen
during three months without apparent loss of binding ability.
Aliquot of the fractions were taken for electron microscopy -
before freezing. The sucrose contained in the suspension
did not interfere with the binding of the labeled HCG.
A~quots of ovarian homogenate and suitable aliquots
of the various fractions obtained from the continuous sucrose ~-
density gradient were solubilized in 1 M. NaOH containing
O.l~ sodium dodecyl sulfate to determine the protein content
* Trade Mark -25-
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10811Zl
- by the method of Lowry (J. Biol. Chem., Vol, 193, page
265, 195l ) using bovine serum albumin as the standard.
Aliquots of the various fractions were also combined
with HCG labeled with ll25 and the degree of binding
measured in each case. On the basis of protein concen-
tration, the plasma membranes containing the specific
receptor for the labeled HCG showed a 10-fold greater
binding than the crude ovarian homogenate.
Each fraction was also analyzed for contamina-
tion by investigating its enzymatic activity. Each frac-
tion was assayed for 5~ Nucleodiase activity by the method
of Song et al. (J. Biol. Chem., Vol 262, page 694, 1967)
and the inorganic phosphate liberated was measured by the
method of Fiske et al. (J. Biol. Chem., Vol. 66, page ~75,
15 , 1925). Cytochrome C reductase was assayed according to the
method of Cooperstein et al. (J. Biol. Chem., Vol. 189, ,
page 665, 195l ).
As a further check as to purity of the plasma
membranes, the ;ndividual fractions ere subjected to electron
microscopy. An aliquot of each protein fr,action was mixed
for 24 hours in 6.25 glutraraldehyde in o.o67 M. cacodylate
buffer of pH. 7.3. The samples were washed for 5 minutes in
chilled 0.25 M. cacodylate or phosphate buffe'r containing
1~ OsO~, of pH. 7.3 for 2 hours. Subsequently, all samples'
were deh'ydrated by passing through a graded series of alcohol
and embedded in either Epon or Araldite. Sections of o.o6-
0.09 um. were cut and stained in a 4~ aqueous uranyl acetate
solution and photgraphed by a,Phillips Em-300 electron micro-
scope.
26 -
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- lV811Z~
A fraction showing the highest binding with the
labeled HCG, a low enzyme activity and containing pure
plasma membranes as shown by electron microscopy was eluted
at a sucrose density of 1.16 (ca 35% w/v). Suitable aliquots
of this fraction were stored in liquid nitrogen and used
as the receptor.
Ill. Procedure of the Radioreceptorassay
The radioreceptorassay was performed in 10 x 75
ml. polystyrene tubes according to the protocol set forth
in Table 1.
Table I
.
Plasma membrane
Sample Diluentlprotein: 40 ug l~5l-HCG
Blank 100 ul 100 ul 100 ul
HCG3(ng/ml)
3.0 100 ul 100 ul 100 ul
6.2 100 ul 100 ul 100 ul
12.5 100 ul 100 ul 100 ul
25.0 100 ul 100 ul 100 ul
50.0 100 ul 100 ul 100 ul
100.0 100 ul 100 ul 100 ul
Plasma (dil.
1:2 to 1:50) 100 ul 100 ul 100 ul
.
10 mM. Tris-HCl buffer of pH. 7.2 containing 0.1~ BSA,
1 mM. CaC12 and 20 I.U. Trasylol per tube.
2Approximately (1.5 ng = 50,000 cpm).
312,000 I.U. per mg. (r7)~
~ - - . - - . ~ - - . . . .. , ., - . . . , . . . - -
- 10~1121
,, ~,
The protocol for simultaneous radioreceptorassay
of hPRL and hCG is shown in Table la.
Table la
_
Unlabeled Hormone Plasma Labeled Hormone3
Membranes2 Control
hPRL hCG l2sl_hpRL 131l-hCG
00 u15 100 u15 1OO Ul 1OO ul 100 ul 50 ul
1OO.O 0.2
50.0 o.4
2S (~ ~ ~
o.8 25.0
o.4 jo.o
0.2 1OO.O ~ / ~ / ~ /
Doubling dilution starting at 100 ng in 100 1 made in
incubation buffer.
2Approximately 100 g protein of plasma membranes isolated
from pregnant cow corpora lutea.
I-hPRL (60-70,000 cpm/tube~ and l-hCG (25-~o,ooo
cpm/tube) made in incubation buffer.
4Plasma from completely hypophysectomized subjects showing
undetectable levels of hPRL and hLH in respective readio-
immunoassays.
Incubation buffer: 10 mM tris buffer, pH 7.2 containing
1 mM MgCl2, CaCl2, 0.1~ (w/v) BSA, and 50 IU of Trasylol.
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lV811Z~
In Table 1, the incubation was performed for 30
minutes at 37C. in a Dunaboff water bath shaker. One ml.
of chilled diluent was then added to each tube. The con-
tents of the tubes were mixed on a vordex mixer and centri-
fuged for 20 minutes at 3,000 xg. in a refrigerated Sorvall
centrifuge (Model RC2B; rotor type HS-4). The supernates
were aspirated and the pellets were counted in a Packard
Autogamma counter with a 51~ efficiency for 1125. The
standard curves and the hormonal concentration in the
plasma samples were calculated by a time sharing IBM com-
puter using logit-log transformations.
A logit-log linearization of the competitive
inhibition response of the readioreceptorassay of HCG is
shown in the figure of drawings attached hereto. The
sensitivity of the assay was less than 3.0ng/ml. with a
precision of +10~ . The intra and interassay variations,
as determined by assaying a plasma pool at various dilutions
in twelve assays, were +5~ and +15~, respectively, through-
out the range of the assay, thus providing evidence of the .-
high reproducibility of the assay. The plasma sample from
a pregnant woman exhibited dose response identical to the
HCG standard, confirming the validity of the assay. There
was a complete lack of cross-reaction with highly purified
preparations of FSH, PRL and HPL, as well as plasma from
hypothyroid subjects, acromegalics and women during post-
partum lactation, thereby indicati~ng a high specificity of the
radioreceptorassay. The radioreceptorassay did not dis-
criminate between HCG and LH; however, a radioimmunoassay
using Il~5 labeled FSH, LH and HCG and antibodies to their
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1 hormone speciFic beta-subunits have shown that there is
no rise in the plasma levels of Lll and ~S~I during early
pregnancy. ~lence, lack of discrimination between HCG
and Ll~ does not effect the detection of pregnancy when
utilizing the readioreceptorassay.
- Binding of l-hPRL to Ovarian Homogenate.
A reaction mi,xture containing 100 ul of incubation
buffer containing 10 IU Trasylol (FB~ Pharmaceuticals, New
York, New York), 50 ul of receptor preparation equivalent to
1-300 ug of protein and 100 ul of l-hPRL (S~; 50-70 uCi/
ug) were incuhated at, 37C. for 2 hours in the absence and in
the presence of 1 ug of unlabeled bovine PRL. ~t the end of
incubation, 1 ml of ice cold buffer was added to all tubes
and tubes were centrifuged at 5,000 rpm for 20 minutes.'
The supernatarlts were aspirated. The pellets containing
receptor bound hormones were counted in an *~utogamma counter
with a 51~ ef~iciency for 1 (Packard Instrument Company,
Downers Grove, Illinois). Specific hinding was calculated
as the difference of binding in the tubes with and without
unlabeled P~,.
Speci~ic binding of l-hPRL to plasma membrane
from bovine corpora lutea was a saturable process. The
saturation was demonstrated by incubation of plasma membrane
from bovine corpora lutea (150 ug protein) at 37C. for 2
;25 hours in 250 ul volume with varying concentration of 1 51-
hPRL. Binding capacity/mg of protein and Kd Calculated by
Schatchard analysis of data obtained from the saturation
curve were 1.4 x 10 m and Kd = 1.4 x 10
*Trade lilark
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.
' ' - :: ' - ' ' ~ - - : :
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S ifi bi di of 1?5 I hPRL to ovarian homo-
genates increased as a function of the amount of homogenates
added and was inhibited by addition of 1 ug unlabeled bovine
PRL to the incubation mixtures. Using 100 ug of homogenate,
approximately 20-30~ of the l25l-hPRL was bound in the
absence of unlabeled PRL.
At 37C., binding was rapid during the first
30 minutes; thereafter, binding increased slowly to reach
an equilibrium at 2 hours. At 0C., little binding was
observed even after 18 hours incubation.
In order to demonstrate the specificity of lZ5l-
hPRL binding to ovarian homogenates, human growth hormone
(hGHl, human follicle stimulating hormone (hFSH), human
luteinizing hormone (hLH), bovine PRL (bPRL), ovine PRL
(oPRL), and human chorionic somatomammotropin (hCS) were
incubated at various concentrations. No displacement of
5l-hPRL bound to receptor protein was observed by hFSH
and hLH. hCS and hGH were capable of displacing receptor-
bound lZ5l-hPRL, however, the potency of hGH preparation
in displacing receptor-bound l25l-hPRL was approximately
0.5-1'~ of hPRL. Bovine and ovine PRL preparations were
shown to be essentially of similar potency in competing with
lZ5l-hPRL. A lack of competition by FSH and LH with PRL for
receptor binding demonstrates PRL specificity for the re-
ceptor sites hCs, which was both structural and biological
similar;ties to PRL showed some cross-reaction; however, the
cross-reactivity of 0.5-1 observed with the hGH preparation
is consistent with reported contamination of hGH to a very
similar degree.
- 31 -
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, . . . ,: "
)811~1
1 IV. Clinical Tests
100 subjects who were overdue for the first expected
menses and were potential candidates for mini-abortion
had blood samples drawn for the radioreceptorassay and 24
hour urine collected for the agglutination test *(Pregnosticon-
Dri-Dot Test). Physical and pelvic examination, change in
uterine size and consistency, and other symptoms of a possible
pregnancy were evaluated. If the radicreceptorassay was
positive, irrespective of a positive or negative Pregnosticon
test, a mini-abortion was performed. All specimens were sent
to the pathological laboratory to confirm the presence of
pregnancy. Three patients aborted spontaneously, and three
who developed ectopic pregnancy will be discussed separately.
The blood samples were centrifuged at 2,500 rpm. at 40C. for
15 minutes, and the plasma or serum was stored at -20C. until
used.
The radioreceptorassay for HCG in blood was performed
77 times for 72 patients to determine the presence of early
gravidity. The accuracy of the test was determined by comparing
it tQ the Pregnosticon-Dri-Dot* test, the presence of gravidity
was confirmed by the histopathological examination of endometrial
specimens obtained at elective or spontaneous abortion. The -
usual clinical criteria, for example, the enlarging uterous
or the onset of menstruation, provided further evidence to
confirm the accuracy of the radioreceptorassay.
Forty-one patients exhibited a negative radiorecep-
torassay, and one false positive Pregnosticon result was
associated with this group. All of the 41 subjects were
confirmed as not pregnant by further clinical observations.
* Trade Mark -32-
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.:
llZ~
In 14 cases of abortion, the pathological
diagnosis of the tissue extracted confirmed the result
of the radioreceptorassay. Seven of the 14 miniabortions
demonstrating decidual or placental tissue, which confirmed
pregnancy, had a positive radioreceptorassay but negative
Pregnosticon test.
; The Pregnosticon test was also not sensitive
enough to detect the early pregnancy, whereas the radio- -
receptorassay was capable of detecting a pregnancy as early
as day four following ovulation and proved of great value
in cases of threatened abortion. Four early threatened
abortions within the first seven weeks of pregnancy demon-
strated positive receptorassays, but at the same time
negative Pregnosticon test. The patients aborted in each
instance spontaneously, and the uterine contents were re-
covered revealing chorionic tissue. In the four pregnancies
which terminated in spontaneous abortion, staining continued
for 10 to 14 days and the uterus was not sufficiently en-
larged for the duration of the gravidity, sugyesting an
abnormality in growth and development of the embryo and
placenta.
From 20 subjects visiting the fertility clinic,
blood samples were drawn daily during the menstrual cycle
for the determination of FSH and LH by radioimmunoassay.
On the basis of BBT, plasma levels of FSH and LH, urinary
excretion of estrogen and progesterone and characteristic
changes in the cervical mucus as~evidence of ovulation,
these women were advised to become pregnant. The blood
samples of four of these women who became pregnant were
- 33 -
.
iO811~1
analyzed to establish the levels of HCG by the radio-
receptorassay and the levels of FSH and LH by radioimmuno-
assay on the day of ovulation and during early pregnancy.
The day of ovulation in these four pregnant women was sub-
sequently confirmed by FSH and LH determination in the
blood. A total of 15 normal pregnancies were tested during
the period encompassing ovulation and early pregnancy.
summary of all of the subjects treated is presented in
Table 11.
Table 11
Days following Radiorecep- Agglutination
No. missed menses tor Assay Blood Test - urine
Miniabortions
3 8-11 + +
8 6-14 +
5x 1-11
Normal Pregnancies
8 14-33 + +
7 0-42 +
Possible Ectopics
1 14 +
Spontaneous Abortion
3 5-17 +
Non-Pregnant
43 0-40 _ +
*5 Non-pregnancies reported twice
- 34 -
..
; ` 1()811Z~
The following are additional case history
summaries which serve to further illustrate the value of the -
radioreceptorassay technique.
A 23 year old para 111, gravida IV was seen
eight days following the missed menstrual period. The
Pregnosticon urine test was negative, and the radioreceptor-
assay was positive. A miniabortion performed on the follow-
ing day demonstrated placental tissue.
A 20 year old nulligravida had an intrauterine
device inserted, and nine months later menses were nine
days late; the radioreceptorassay was positive. This assay
was again positive two days later and a miniabortion re-
vealed chorionic tissue. Two Pregnosticon tests performed
on the same days were negative.
; 15 - A 28 year old nulligravida with a twenty-eight
day cycle was seventeen days late with the menses; the
radioreceptorassay was positive and the Pregnosticon test
was negative. Several days later a spontaneous abortion
occurred. Three months later she was seen again five days
after the first missed menstrual period. Again the radio- -
receptorassay was positive and the Pregnosticon test was
negative. The uterus failed to grow, black staining began
~ and persisted until a spontaneous abortion reoccurred four
- weeks after the missed cycle. Tissue examination of the
second abortion revealed an amniotic sac without fetus. ~
In another instance, a 30 year old gravida I ~-
was seen twelve days after a missed cycle. At this time,
the radioreceptorassay was positive and the Pregnosticon
test was negative. After two weeks of irregular staining
and questionable uterine enlargement, spontaneous abortion
occurred.
- 35 -
lOt~ilZ~
In two patients with suspected ectopic pregnancy,
one showed a positive radioreceptorassay and the other a
negative test. The former was confirmed to have an un-
ruptured tubal pregnancy. Both had negative Pregnosticon
tests. The following is a brief account of the ectopic
pregnancy: A 40 year old para 1, gravida I missed one
menstrual cycle and immediately thereafter developed mild
lower abdominal pain and vaginal spotting which per-
sisted for two weeks. The hemagglutination inhibition
tube test was negative on the day following admission.
The radioreceptorassay on the second day was positive.
On the third day, currettage revealed no evidence of
pregnancy; cul-de-sac puncture was negative, laparoscopy
demonstrated an unruptured left ectopic which was removed
at subsequent laparotomy.
Thirteen patients were admitted to the hospital
because of suspected ectopic pregnancy. The last menstrual
period ranged from 23-76 days prior to admission. Lower
abdominal pain, amenorrhea, frequent vaginal staining, and
an adnexal mass were the usual findings. Plasma samples
were obtained from each patient prior to surgery and in
one instance on four separate days for the radioreceptor-
assay of HCG. Conventional hem- or latex agglutination
tests were performed on urine samples at the same time.
The results of pregnancy tests and pathological finds are
presented in Table 111.
Table 111 indicates the results obtained by the
radioreceptorassay and hemagglutination test in 13 suspected
ectopic pregnancies, and in addition, the time from the last
menses and the tubal histological finds. Seven of the 10
ectopic pregnancies had a positive radioreceptorassay and
negative hemagglutination p-egnancy test. One false positive
, .
- 36 -
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hemagglutination test and no false positive radioreceptor-
assays were obtained. There was a wide range for the dura-
tion of pregnancy spanning between 23 and 76 days past the
last missed period.
The logit-log transformation of standard HCG
dose-responsive curve yielded a sensitivity of 0.3 ng or
3 mlU/HCG/ml (Fig. 2). In this figure, TSH is the legend
for plasma from hypothyroid patients; HGH, plasma from
acromegalic subjects; PRL, plasma from women with post-
partum lactation. mlU of 2nd International Reference
Preparation equivalent to corresponding ng of HCG are indi-
cated on the ordinate. Various dilutions of the plasma
sample from a pregnant woman yielded a slope parallel to
that of HCG indicating the validity of the assay. There
was no cross-reaction with FSH, TSH, HGH, and HPRL in the
assay. There was a 98-102% recovery of HCG added to the
plasma samples of known hormonal concentration. The intra-
and inter-assay variation in the estimates of hormonal --
levels of plasma pools was 6~ and 11~" respectively. The
radioreceptorassay, however, did not discriminate between
LH and HCG. This drawback was circumvented by the obser-
vations 1) that neither LH nor FSH rise during early preg-
nancy as determined by specific ~ subunit radioimmunoassay
of FSH, LH, and HCG in the blood, 2) that the standards
contain the same amount of plasma obtained from non-preg-
nant women during luteal phase containing low levels of LH,
3) that all unknown samples are compared with a pool of
plasma from non-pregnant and various dilutions of a pool
of plasma from pregnant women analyzed routinely with each
assay for accuracy and quality control, and 4) finally, that
during early pregnancy the HCG-LH levels are 2-3 fold higher
than basal LH levels in non-pregnant women during the luteal
phase.
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,
iO~112~
In Fig. 3, the HCG levels in 10 ecotopic preg-
nancies are compared with normal intrauterine gravidity
in 2 subject (H.D., R.S.) and 1 induced ovulation and ~ -
pregnancy (S.L.) with 10,000 IU of HCG which was monitored
with radioreceptorassay. The plasma levels of HCG during
early induced or natural pregnancy were similar. The day
of pregnancy was based on the last menstrual period re-
ported by the patients. In one of the 10 patients (A----A),
the readioreceptorassay was performed on days 21, 24, 30,
and 42 of pregnancy. The positive radioreceptorassays on
days 21 and 24 were associated with negative Pregnosticon
tests. The onset of the HCG rise following LH peak was
first detected in the blood samples obtained first on day
6-8 following ovulation and it might be earlier following
fertilization. Three of the ectopics had assays performed
prior to the first missed menses and the levels of HCG in
4 were in the range of the normal pregnancy. The other
6 ectopic HCG levels were lower than the norma ! . These
observations suggested that the early nidations in the tube,
before rupture and hemorrhage, could secrete normal quanti-
ties of HCG. Later, however, when hemorrhage, increased
separation and reduced blood supply occurred, the HCG
secretion leveled off.
As shown in Fig. 3, in the present series of 13
suspected ectopic pregnancies, the HCG was 22 and 35 ng/ml
or 0.26 and 0.42 IU, respectively, in two of the 13 cases.
The detection of HCG at these low levels aided in the correct
management of the patient at an earlier stage. The remaining
patients had values of ~00 ng/ml. The levels of HCG on
days 21, 24, 30, and 42 of pregnancy (Fig. 3) were obtained
from the same patient. The initial test was associated with
39 -
- ~ .
'
,
- lUBllZ~
a negative Pregnosticon-~ test; whereas both tests were
positive on three subsequent determinations. On the 64th
day, abdominal intervention was required because of a rup-
tured right ectopic pregnancy.
SUMMARY
The radioreceptorassay of HCG with a sensitivity
of 50 pg or 3 mlU/ml plasma has provided close to 100~'?,
reliability to detect pregnancy following the first missed
cycle. This test performed within 1 hour is ideally suited
for clinical detection of ectopic pregnancy especially in
patients who require immediate surgical intervention.
Thirteen patients with suspected ectopic pregnancy were
- evaluated by the radioreceptorassay, one of which was followed
with four separate determinations. The results of the assay
were subsequently compared with those of hemagglutination
pregnancy tests, clinical symtoms, and pathological findings.
All the patients were diagnosed accurate1y by the radio-
receptorassy, even when hemagglutination tests yielded
false indication of pregnancy. The HCG levels during ectopic
pregnancies by this assay are generally lower than those
during a normal intrauterine pregnancy; in addition, much
earlier (prior to the rupture) detection of pregnancy is
possible than by hemagglutination tests. Furthermore,
ectopic pregnancy may be excluded in patients admitted to
the hospital with acute abdominal emergencies. ~
; 40 ~ -
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