Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
20856 JAW
IMMUNOASSAY FOR HUMAN CHORIONIC GONADOTROPIN
The invention described herein was made in the course of
work under grant numbers HD~15454 and RR-00645 from the
National Institutes of Health, United States Department
of Health and Human Services, U.S.A.
Background of the Invention
Throughout this application various publications aurora-
furred to to provide background information useful for a
complete understanding of the invention.
Immunoassay for human chorionic gonadotropin (hug) are
known and have been widely used, particularly in the
diagnosis of pregnancy. Recent advances in immunology
involving hybridomas end mo~oclonal antibodies have
greatly increased the sensitivity of previous immune
assays. See, for example, Ehrlich, PI et at., Journal
: of Immunology 128:2709 (1982); Wade, HUG. et at., Olin.
Chum., 2~:1862 (1982~; Shims, STY. et at., Olin. Chum.,
28:546 (1982); and Petters son, K. et at., Olin. Chum.,
29:60 (1983). Serum-derived antibodies have also been em
plowed. See, for example, Seiko, T. et at., Act Endow
crinologica 97:562 (1981), Allah, AIR. et at., J. Clinical
Endocrinology and Medicine, 47:767 (1978) and.Wehmann,
RYE. et at., Amer. J. of Obstetrics and Gynecology,
140:753 (1981). Immunoassay involving serum-derived an-
tibodies have included antibodies directed to the unique
car boxy terminal portion of the subunit of hug (ibid.;
and irked S. et at., Endocrinology, 110:1555 [owe
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Although previous improvements in hug immunoassay have
provided increased sensitivity or specificity, or both,
it has nut been possible until the present invention to
obtain the greatly enhanced sensitivity and almost Abe
solute specificity achieved using the immunoassay disk
closed and claimed herein. This new immunoassay permits
detection of smaller increases in hug levels, and detect
lion much sooner after insemination, than previously
possible. It also permits hug measurements in cancer
diagnosis unimpaired by significant huh cross-reactions.
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Summary of the Invention
This invention concerns an immunoassay for detecting hug
or measuring the level of hug in a sample. The assay
includes an antibody directed to the car boxy terminal
portion of the subunit of hug and a monoclonal antibody
directed to a determinant on hug at a locus sufficiently
remote from the car boxy terminal portion of the subunit
of hug that both antibodies can simultaneously bind to
hug, wherein at least one of the antibodies is detectable
when both are bound to hug.
In a presently preferred embodiment the immunoassay can
deject or measure levels of hug or hug in a urine sample
and includes a detectable, purified, serum-derived anti-
body directed to the car boxy terminal portion of the
subunit of hug and a matrix-bound monoclonal antibody
: directed to a locus on the subunit sufficiently remote
from the car boxy terminal portion that both antibodies can
simultaneously bind to ho
The assay may be used to detect hug or to measure hug
levels in urine samples. In this way the presence of hug-
producing neoplasms, subclinical spontaneous abortions
and ectopic pregnancies can be diagnosed.
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Brief Description of the Drawings
Fig. 1 is a standard curve showing use of the assay to
measure hug in buffer I) and in urine (~---~) and
to show extent of cross~reac~ivity with huh in urine
(I ---- En ) .
Fig. 2 illustrates the sensitivity of a conventional radio-
immunoassay (Con A Assay, 1 as compared with the newly
developed sandwich assay (~---~) in the measurement of hug
levels in an artificially inseminated female patient. Also
depicted is the hypothetical pattern of hug levels occurring
as a result of subclinical spontaneous abortion (----).
I
_ 5 3
Recent improvements in immunoassay technologies, par-
titularly the development of monoclonal antibodies, have
permitted improvements in the sensitivity of assays for
human chorionic gonadotropin (hug). However, until the
present invention assays have not been available which
permit detection of increases in hug levels a few days
after insemination and which posses essentially absolute
specificity for hug, having no cross reactivity with huh.
Specifically, this invention provides an immunoassay fox
detecting hug or measuring levels of hug, or both, in a
sample. The immunoassay includes an antibody directed to
the car boxy terminal portion of the subunit of hug which
is a unique peptize sequence not found in huh. The
immunoassay also includes a monoclonal antibody directed
to a determinant on hug at a locus sufficiently remote from
the car boxy terminal portion of the subunit that both
antibodies can simultaneously bind to hug. Finally, at
least one of the antibodies is capable of detection when
both antibodies are bound to hug.
Although in principle any antibody directed to the car boxy
terminal portion of the subunit of hug would be effect
live, the presently available and preferred antibodies
are purified, serum-derived antibodies. Examples include
R525 and R529 as described more fully hereinafter. Dow-
ever, the present invention is not limited to serum-
derived antibodies and also encompasses monoclonal anti-
bodies directed to the car boxy terminal portion of the
subunit if and when efforts to produce such monoclonal
antibodies are successful.
-- 6 --
~L2~5~9
Either antibody or both antibodies are capable ox detect
lion when the antibodies are bound Jo hug. Various means
for rendering an antibody detectable are known to those
skilled in the art. Merely by way of example, suitable
means include radioactive labeling, e.g., 125I labeling,
fluorescent labeling or linkage to an enzyme which cat-
Allis a detectable reaction, that is, an LISA approach,
ego, linkage of an antibody to horseradish peroxides.
An additional approach involve the use of a third anti-
body directed to one of the antibodies of the assay worth third antibody, either serum-derived or monoclonal,
it detectable, e.g., labeled with a radioactive isotope or
a fluorescent moiety.
The immunoassay of the present invention may be carried
out totally in liquid phase, totally in solid phase or in
a mixed liquid/solid system. Presently it is preferred
what one of the antibodies, preferably the monoclonal
antibody directed to a locus remot@-from the arks
terminal portion of the subunit be attached to a solid
matrix, go agrees or Suffers.
Although the immunoassay may be used with either urine,
plasma serum or other samples, it is particularly
desirable to employ the immunoassay on urine samples
because of the greater ease and convenience of obtaining
such samples. This is, in fact, one ox the substantial
advantages provided by the present invention since most
presently available immunoassay for hug are not suitable
for use with urine samples.
The presently preferred embodiment of the invention in-
voles an immunoassay for detecting hug or measuring
levels of hug and thereby detecting or measuring hug.
I This immunoassay includes a purified, serum-derived anti-
. ,~. ;,
' ':` ,1
_ 7 _ 3
body directed to the car boxy terminal portion of the
subunit of hug and a monoclonal antibody directed to a
locus on the subunit sufficiently remote from the car boxy
terminal portion that both antibodies can simultaneously
bind to hug. In the preferred embodiment the purified,
serum-derived antibody is detectable when both antibodies
are bound to hug alone or as hug, and the monoclonal
antibody is attached to a solid matrix.
This invention also concerns a purified antibody to the
car boxy terminal portion of the subunit ox hug, prey-
drably labeled or otherwise detectable, e.g., isle-
bleed.
The immunoassay ox this invention provides a method of
detecting hug or measuring the level of hug, or both, in
a urine sample. The sample to be tested is placed in the
immunoassay under suitable conditions permitting format
lion of a detectable or measurable complex with hug. The
resulting complex may then be detected if present or the
level of hug present determined by reference to a standard
containing a known amount of hug.
Measurements of hug levels in turn can be used in the
diagnosis or identification of disorders which involve
production, or elevated levels, of hug. Examples of such
disorders include certain neoplasms, e.g., male testicu-
far cancer, subclinical spontaneous abortions and ectopic
pregnancies.
Methods for utilizing immunoassay for such purposes are
well known to those skilled in the art and therefore are
not described hereinafter in greater detail.
The following section entitled "EXPERIMENTAL DETAILS" is
8 ~49
set forth to aid in an understanding of the present
invention but is not intended, and should not be con-
stropped, to limit the invention as defined by the claims
which follow thereafter.
US
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EXPERIMENTAL DETAILS
Materials and Methods
Reagents
The antibodies used in this experiment were R525 and R529,
Birken, S. en at., Endocrinology, 110:1555(1982), rabbit
antisera directed against the hug CUP d~terminan~s, and
B101, Ehrlich, PI et at., Journal of Immunology,
128:2709 (1982), a mouse monoclonal antibody directed
against an hug confirmational determinant. The methods
of preparation and characteristics of these antibodies
were as published in the preceding two cited references.
The preparation of highly purified hug and hug subunit
have been previously described. Can field, RYE. and
Morgan, F.J., in: Methods in Investigative and Diagnostic
Endocrinology, edited by Benson, SPA. and Yule US
North-Holland, Amsterdam, p727 (1974). Human luteinizing
hormone was a gift from the National Pituitary Agency
university of Maryland School of Medicine) NOMAD, NIX.
Concanavalin A covalently linked to agrees (Con A
Suffers), an hydrous a-methyl-D-mannoside and bovine y-
globulin, Cohn fraction II were purchased from Sigma Co.
Cyanogen bromide (CnBr) activated Suffers 4B was ox-
twined from Pharmacia Fine Chemicals and DEE Affi-Gel
Blue from Byrd Laboratories. The preceding purchased
materials were used in accordance with the manufacturers'
instructions
Monoclonal Antibody production in Auschwitz Fluid,
Purification and Coupling to Sepharose~4B
The production of monoclonal anti-hCG~ was amplified
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utilizing the mouse Auschwitz tumor system. Lola, H. and
Brooks, D. in: Monoclonal Hybridoma Antibodies: Tech-
piques and Applications, edited by JAR Harley, CRC
Press, Inc., Bock Rayon, Florida, p50 (1982). One million
Blue producing hybridoma cells were injected into each of
thirty CD2Fl strain mice (west Seneca Laboratories) two
weeks after they had been primed with one-half ml of
pristine. Collection of Auschwitz fluid commenced three
weeks after injection of the cells and continued for two
month. A total of 330 ml of Auschwitz fluid was pooled and
stored at -80C.
One Alcott of forty-six mls was dialyzed extensively
again 0.025 M Tris-HClg pi 7.5, containing 0.05 M Nail
and 0.02% sodium aside at 4C. The dullest was applied
to a 25 x 300 mm column of DEE Afi-Gel Blue and eluded
in five ml fractions with the same buffer used for dial-
yip One hundred fractions containing the peak absorb-
ante of 280 no were pooled. The total amount of protein
recovered in the preparation was calculated to be 1.46 g
using the method of War burg and Christian. War burg, O. and
: Christian, W., Become. Z., 31OJ384 (1941). Electron
pharisees of the preparation on non reducing SDS polyp
a rylamide gels, Weber, K. and Osborne M., J. Blot. Chum.,
2 :4406 (1969), indicated that at least 80% of the
protein content was globulin.
Forty mls of the partially purified Auschwitz preparation
containing-approximately 80 my globulin was dialyzed
against O .1 M Nikko, pi 8.0, containing 0.5 M Nail at 4C.
The dullest was then coupled to ten grams of CnBr
activated Suffers 4B according to the manufacturer's
instructions. A 50% suspension Volvo of B101 coup
pled Suffers 4B was prepared in 10 my phosphate buffered
US saline, pi 7.4, containing 10 my Nudity, 0.1% sodium
aside and 0.1% bovine globulin (buffer B) and stored at
4C.
S
and Iodination
Ten mugs of ho was coupled to two grams of CnBr activated
Sirius 4B. The hug coupled Suffers 4B was used to
form an 0.9 x lo cm column which was used for affinity
purification of R525. A 54 ml pool of R525 from several
different bleeds between boosting was recycled o'er the
column continuously for 30 hour at 4C using a peristaltic
pump. The column was washed extensively with saline to
lo remove loosely adhering material. The column was then
eluded successively with 20 ml of EM guanidine-HCl, pi
3.0, and 20 ml of 6 M guanidine-HCl, pi 3Ø 'rho effluent
was collected in one ml fractions and the absorbency at 280
no determined. The absorbency profile had two discrete
protein peaks corresponding in position to the areas of
elusion with 3 M and 6 M guanidine-~Cl~ The fractions
having the peak absorbs at 280 no were pooled and then
dialyzed extensively against lo my sodium acetate, pi 5.5,
followed by 0.3 M Nope, pi 7.5, at 4C. The dialyzed
preparations, an Alcott of the unpurified R5Z5 pool and
also the R525 pool which had been circulated over the hug
affinity column were tested for their ability to bind
5I-hCG. Only the unpurified R525 pool and the purify-
cation product eluded from the column with S M guanidine-
Hal had significant binding. A 1:1,500 dilution from a
total volume of 5.7 ml of the purified R525 bound 50% of
the trade as opposed to a 1:600 dilution of the unpurified
antiserum pool. The total protein content of the purified
R525 was calculated to be 1.9 my. War burg, O. and
Christian, W., Become. Z., 310:384 (1941).
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Thirty of the purified Russ radio labeled with lmCiNa
125I (Americium) using Iodogen (Pierce Chemical Company)
as the oxidizing agent. Franker, PI and Speck, Jo, JO
Become. Buffs. Research Comma., 80~849 (1978)~ The
specific activity of the iodinated product was approxi-
mutely 20 swig protein.
Conduct of Sandwich Assay
The conditions for the sandwich assay described below are
the result of optimization by detailed analysis of reagent
concentrations, incubation times and temperatures no-
squired to jive maximum hug binding. Prior to assay, urine
samples were adjusted to pi 7.4 with Noah and centrifuged
at 3000 x g for 15 minutes. Duplicate or triplicate four
ml allocates of urine, standards containing 0.004-0.5 my
hCG/ml in Buffer B, or Buffer B alone (for determination
of nonspecific bindings; binding of trace IRE] in
the absence of hug) were pupated into 12 x 75 mm polyp
styrenes tube. Two tenth ml of a 6.25% suspension of B101
coupled Suffers 4B in Buffer B, containing approxi-
lately 25~g y-globulin, was pipette into each tube. The
tubes were capped, placed horizontally on a Lab quake
Shaver (Lab industries) and incubated for two hours at room
temperature with shaking in order to extract hug from the
samples. The tubes were centrifuged for 15 minutes at
3,000 x g. The supernatants were removed by aspiration,
and the Bl~l-Sepharose 4B pellets washed 2x with 2 ml
suffer B 1% Winnie 20.
One-~enth ml of Buffer B containing 50,000 CAM IRE
was added to each tube. The samples were incubated
vertically for 48-72 hours at 4C with shaking on the
LabquakP Shaker. The tubes were then washed three times
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with 2 ml of Buffer B I Tweet 20 to reduce nonspecific
binding.
The radioactivity remaining after washing was determined
in a Packard Auto-Gamma Scintillation Spectrometer. Data
reduction for the generation of standard curves was act
complished using a four parameter logistic fit. Rod bard,
Do and Hull, Do Pro Radiomen-
as sand Related Procedures in Medicine, International
Atomic Energy Agency, Vienna, Austria, Unipub., New York,
ply (1974).
.
the hug radioi~munoassay was conducted using the R529
antiserum to the hug CUP determinants to measure hug
extracted from urine with concanavalin A as described by
Wyman et at. Wyman RYE., et at., Clinical Chemistry,
_:1997 (1981)~
Patients
First morning voided and random urines collected by normal
volleyers at the Columbia Presbyterian Medical Center
were used in order to establish the normal range of hug
excretion. The measurement of hug for detection of early
pregnancy was conducted using f first morning voids pro-
tided my patients in the Department of Obstetrics and
Gynecology, Columbia College of Physicians and Surgeons
these were normal women who were artificially inseminated
because of the male partners' infertility.
In order to correct for differences in the specific
~Z~5~
gravities of the various urines collected, urinary hug
concentration was noxmali~ed to cxeatinine concentration
Ryes
Sandwich Assay Standard Curve
A typical standard curve generated for hug binding in the
sandwich assay is shown in Fig. 1. The hug dosages which
give binding equivalent to 10% and 90~ of Max (ED lo and
Ego) have been tentatively accepted as the limits of the
usable range of the standard curve. In the assay demon-
striated ED lo and Ego correspond Jo 0.01 and 0..50 no
hCG/ml, giving a fifty fold usable range. The Nonspecific
winding NUB in the assay is reduced to an acceptable
level by extensive washing of the pellet with buffer
containing Tweet 20 and bovine y-globulin. In the assay
represented NUB way 2.5% of the total counts added.
Fig. 1 also shows the dose response curve of hug extracted
from normal male urine which had Boone spiked with dosages
identical to those used in the standards. The dose
response curve for hug in urine is essentially identical
to that for hug in Buffer B. The slope and Educe of the dose
response curve were 1.173 and 9.066 ng/ml for hug in urine,
compared to 1.098 and 0.072 ng/ml for hug in buffer.
The dose response curve for ho added to normal male urine
is alto shown in Fig. 1. It should be noted that the units
for huh concentration are~g/ml as opposed to ng/ml for hug
concentration. A concentration of 2~g hLH/ml is required
to obtain a dose response equivalent to 0.01 no hCG/ml.
Thus, the cross-reactivity of this concentration of huh in
the sandwich assay is 0.0005% on the basis of mass. This
\
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I
level of huh is approximately nifty fold higher than those
present in urine from women at the mid-cycle huh surge.
Saxon, BOB. et at., Fertility and sterility, 28:163
(1977). Therefore, physiologic concentrations of huh are
incapable of interfering in the sandwich assay.
In summary, Fig l shows thaw the assay is equally effect
live for the measurement of hug in buffer and in urine,
highly sensitive, ire., capable of detecting hug at levels
down to 0.004 ng/ml and that cross-reactivi~y with ho is
exceptionally low, i.e., about 0.0005~, thereby rendering
he assay absolutely specific for hug 7
Fig. 2 shows the use of the assay to measure hug levels in
15 the urine of a artificially inseminated female patient at
various days after insemination. By way of comparison
Fig. 2 also show the use of a standard radio immunoassay
to measure hug levels in the same patient's urine. As is
clearly indicated in Pig. 2, the assay of the present
20 invention is capable of detecting elevated hug levels
after the sixth day following insemination. By contrast,
the standard RIP (sandwich assay) is less sensitive and
cannot detect hug until the tenth day following inseam-
nation.
Fig. 2 also illustrates the application of the assay in the
diagnosis of subclinical spontaneous abortion. The hype-
thetical pattern illustrated in Fig. 2 shows that the
standard RIP is incapable of detecting either the rise or
30 the fall of hug as the result of subclinical spontaneous
abortion while the assay of the present invention will be
able to do so because of its much greater sensitivity.