Ex vivo product of conception test to determine abortion

The invention provides a method for determining the presence of products of conception in a sample derived from the uterus during a D&C, or a therapeutic or spontaneous abortion, and comprises determining the presence in the sample of a fetal restricted antigen, which is found in products of conception but not found in significant amounts in maternal plasma or serum. Since the fetal restricted antigen is not present in significant quantities in maternal plasma or serum, the methods of this invention are reliable even when the sample is contaminated with maternal blood. One fetal restricted antigen is fetal fibronectin. In one embodiment of this invention, the sample is contacted with an insoluble support to which anti-(fetal restricted antigen) antibody is adhered, and the fetal restricted antigen binding to the support is determined. Alternatively, an antibody which binds a class of substances of which the fetal restricted antigen is a member can be substituted for either the capture antbody or the sandwiching antibody, and binding of fetal restricted antigen is determined. Competition assay procedures can also be used. Reagents and reagent kits are included.

FIELD OF THE INVENTION 
This invention relates to methods, reagents and kits for detection of ex 
vivo products of conception. In particular, this invention is directed to 
the determination of products of conception in uterine tissue expelled or 
removed from the uterus through spontaneous or therapeutic abortions. 
BACKGROUND OF THE INVENTION 
Determination of the presence of ex vivo products of conception in uterine 
tissue removed in therapeutic or spontaneous abortion is critically 
important to confirm the existence of uterine pregnancy and the 
termination thereof, and to rule out the presence of an ectopic pregnancy. 
If levels of fetal associated antigens in maternal serum or urine indicate 
pregnancy, and the uterine tissue removed during a therapeutic abortion 
contain no products of conception, a possible ectopic pregnancy is 
indicated. Presence of products of conception in uterine discharge 
associated with indicators of spontaneous abortion confirms the abortion, 
while the absence thereof indicated a continuation of pregnancy. 
Usual immunoassay methods for determining the presence of fetal associated 
antigens in uterine tissue samples are not reliable for indicating the 
presence of products of conception since these samples typically contain 
maternal blood. Nonrestricted fetal antigens are usually present in 
maternal blood as well as in fetal and placental tissue. 
DESCRIPTION OF THE PRIOR ART 
Traditional methods for differentiation of true and false labor is often 
difficult before the uterus has contracted sufficiently to produce 
demonstrable cervical effacement or dilatation. Other traditional signs 
and symptoms indicating risk of preterm delivery are passage of cervical 
mucus, often slightly bloody, low backache, pelvic pressure due to descent 
of the fetus, menstrual-like cramps, and intestinal cramping with or 
without diarrhea. The traditional approaches are described by Stubbs, T. 
et al, Am. J. Obstet. Gynecol. 155:829-834 (1986). These procedures are 
not applicable or highly unreliable in confirming spontaneous abortion in 
the first trimester. 
Several tests have been reported for diagnosis of preterm rupture of 
membranes. Rochelson, B. et al, Obstet. Gynecol. 69:163-165 (1987) 
reported a rapid, colorimetric monoclonal alpha-fetoprotein (AFP) antibody 
test, based on the presence of high levels of AFP in amniotic fluid. 
However, in the case of bloody specimens, a situation most similar to 
tests of expelled uterine tissue during spontaneous or therapeutic 
abortion, the AFP test was reported to be unreliable since maternal blood 
was frequently the source of the AFP. Huber, J. et al, Obstet. Gynecol. 
80:1183 (1983) reported that a quantitative AFP radioimmunoassay in the 
diagnosis of premature rupture of membranes gave too much overlap with 
nonruptured controls. 
Matsuura, H. and Hakomori, S., Proc. Natl. Acad. Sci. USA. 82:6517-6521 
(1985) describe the discovery of a fetal fibronectin which binds 
preferentially with a monoclonal antibody FDC-6, and that this antibody 
does not bind with normal adult fibronectins. The fetal fibronectin was 
found in placenta, amniotic fluid and fetal connective tissue, as well as 
a number of tumors. The antibody did not bind with constituents of normal 
adult plasma and adult tissue. Structural differences between the fetal 
fibronectin and adult fibronectins were described. Other references 
relating to this discovery are Ali, I. et al, J. Biol. Chem. 256:7671-7677 
(1981); Wagner, D. et al, J. Biol. Chem. 256:11798-11715 (1981); 
Dekiguchi, K. and Hakomori, S., J. Biol. Chem. 258:3967-3973 (1983); 
Hayashi, M. et al, J. Biol. Chem. 256:11292-11300 (1981); Atherton, B. et 
al, Cell. 25:133-141 (1981); Ruoslahti, E. et al, Int. J. Cancer. 
27:763-767 (1981); Zhu, B. et al, J. Biol. Chem. 259:3962-3970 (1984); 
Cossu, G. et al, J. Biol. Chem. 258:5603-5607 (1983); Murayama, K., 
Hakomori, S. et al, Glycoconjugate. 1:155-169 (1984); Teng, M. et al, J. 
Cell. Biol. 80:784-791 (1979); Liu, M. et al, Proc. Natl. Acad. Sci. 
82:34-37 (1985); Kiyotoshi, S., Hakomori, S. et al, Biochem. Biophys. Res. 
Comm. 116:534-540 (1983); Sekiguchi, K., Hakomori, S., et al, J. Biol. 
Chem. 260:5105-5114 (1985); Zardi, L., et al, Int. J. Cancer, 25:325-329 
(1980); and Nakabayashi, H. et al, Cancer Res. 42:3858-3863 (1982); Dot, 
I. Gann. 67:1-10 (1976). Kuusela, P., et al, Scand. J. Immunol. 12:331-337 
(1980) also discloses a monoclonal antibody binding preferentially with 
amniotic fluid fibronectin (fetal fibronectin) and a method for preparing 
the hybridoma therefor. 
A representative listing of known nonrestricted fetal antigens and known 
antibodies which bind selectively or preferentially therewith are set 
forth in PREGNANCY PROTEINS: BIOLOGY, CHEMISTRY AND CLINICAL APPLICATION. 
Grudzinskas, J. et al (editors), New York: Academic Press (1982) and the 
publications cited therein, the entire contents of which and the 
publications cited therein being hereby incorporated by reference in their 
entireties. Examples of identified nonrestricted fetal antigens are 
chorionic gonadotropin (hCG), human chorionic thyrotropin (hCT), human 
placental lactogen (hPL), Schwangerschafts-spezifizisches glykoprotein 1 
or pregnancy specific .beta..sub.1 -glycoprotein (SP1), 
pregnancy-associated plasma protein A (PAPP-A), pregnancy-associated 
plasma protein B (PAPP-B), heat-stable alkaline phosphatase (HSAP) (S, I, 
and F phenotypes), cystine aminopeptidase (CAP), placental protein 5 
(PP5), placenta specific .alpha..sub.1 -microglobulin (PAMG.sub.1), 
placenta specific .alpha..sub.2 -microglobulin (PAMG.sub.2), pregnancy 
associated .beta..sub.1 -macroglobulin (.beta..sub.1 -PAM), pregnancy 
associated .alpha..sub.2 -macroglobulin (.alpha..sub.2 -PAM), human 
chorionic luteinizing hormone-releasing factor (hCLRF), human chorionic 
thyrotropin-releasing hormone (hCTRH), and human chorionic growth 
hormone-releasing inhibiting hormone (somatostatin), all of these being 
fetal proteins which are produced by the placenta, and which have been 
purified and well characterized. These nonrestricted pregnancy antigens 
are compounds and substances found in significant amounts in the maternal 
plasma and serum and are not fetal restricted antigens. 
Isolation and diagnostic examination of fetal cells from cervical samples 
obtained by lavage of the uterine cavity is described in U.S. Pat. No. 
4,675,286. 
An immunological method for determining total fibronectin in samples is 
described in Japanese Patent Application 60091264 (DIALOG database file 
351, WPI Acc. No. 85-161617/27). A non-immunological method is described 
in USSR Patent Application No. 1107051 (DIALOG database file 351, WPI Acc. 
No. 85-055390/09). Separation of total fibronectin (also identified as 
.alpha..sub.2 -sb-glycoprotein, cold-soluble protein and LETS-protein) by 
affinity chromatography is described in U.S. Pat. No. 4,325,867. ATCC HB 
91 (American Type Culture Collection, Rockville, Md.) is a hybridoma clone 
which produces an anti-(cellular and plasma fibronectin) antibody. 
SUMMARY OF THE INVENTION 
The method of this invention for determining the presence of products of 
conception in a sample of tissue suspected to have been drawn from the 
uterus, i.e., derived from the uterus during a dilation and curettage, or 
therapeutic or spontaneous abortion, comprises determining the presence in 
the sample of a fetal restricted antigen (found in products of conception 
but not found in significant amounts in maternal plasma or serum). Since 
the fetal restricted antigen is not present in significant quantities in 
maternal plasma or serum, the methods of this invention are reliable even 
when the sample is contaminated with maternal blood. One fetal restricted 
antigen is fetal fibronectin. 
In general, immunoassay methods comprise interacting the fetal restricted 
antigen in a sample with an anti-(fetal restricted antigen) antibody for a 
time sufficient to permit antigen-antibody binding to occur; and 
determining the presence of said binding. In one sandwich immunoassay 
embodiment of this invention, the sample is contacted with an insoluble 
support to which anti-(fetal restricted antigen) antibody is adhered for a 
time sufficient to permit antigen-antibody binding to occur, and the 
sample is removed from the support. The insoluble support is then 
contacted with an antibody which binds with the fetal restricted antigen 
or the fetal restricted antigen class (i.e., a secondary antibody) for a 
time sufficient to permit antigen-antibody binding to occur, and the 
unbound secondary antibody is removed from the support. The presence of 
secondary antibody on the insoluble support is then determined. The 
secondary antibody can have a physically detectable label which can be 
measured directly on the insoluble support. Alternatively, the secondary 
antibody can be unlabeled, and the secondary antibody can be determined by 
contacting the insoluble support with a labeled antibody which binds 
preferentially with the secondary antibody (i.e., a tertiary antibody), 
removing unbound labeled tertiary antibody from the support, and 
determining the presence of the label on the insoluble support. 
In an alternate sandwich immunoassay embodiment of this invention, the 
sample is contacted with an insoluble support to which is adhered an 
anti-(fetal restricted antigen class) antibody for a time sufficient to 
permit antigen-antibody binding to occur, and removing the sample from the 
support. The insoluble support is then contacted with an anti-(fetal 
restricted antigen) antibody for a time sufficient to permit 
antigen-antibody binding to occur, and removing unbound anti-(fetal 
restricted antigen) antibody from the support. Finally, the presence of 
anti-(fetal restricted antigen) antibody on the insoluble support is 
determined. The anti-(fetal restricted antigen) antibody can have a 
physically detectable label, in which event, the label adhering to the 
insoluble support can be determined. Alternatively, anti-(fetal restricted 
antigen) antibody can be unlabeled, and the insoluble support can be 
contacted with a labeled tertiary antibody which binds preferentially with 
the anti-(fetal restricted antigen) antibody, the unbound labeled antibody 
is removed from the support, and the presence of the label on the 
insoluble support is determined. 
Competition embodiments of this invention using labeled reagent fetal 
restricted antigen comprise contacting a mixture of the test sample and 
labeled reagent fetal restricted antigen with an anti-(fetal restricted 
antigen) antibody adhered to an insoluble support, and determining the 
amount of label which either binds with the insoluble support or remains 
in the solution phase. 
Competition embodiments of this invention using labeled anti-(fetal 
restricted antigen) antibodies can be of more than one type. One 
embodiment using anti-(fetal restricted antigen) antibody bound to the 
insoluble support comprises contacting a mixture of the sample and labeled 
anti-(fetal restricted antigen) antibody with anti-(fetal restricted 
antigen) antibody adhered to an insoluble support, and determining the 
amount of label which either binds with the insoluble support or remains 
in the solution phase. Another embodiment uses reagent fetal restricted 
antigen bound to the insoluble support, and comprises contacting a mixture 
of the sample and labeled anti-(fetal restricted antigen) antibody with a 
fetal restricted antigen adhered to an insoluble support, and determining 
the amount of label which either binds with the insoluble support or 
remains in the solution phase. 
Ex vivo product of conception testing reagents include insoluble supports 
to which are adhered reagent fetal restricted antigen or anti-(fetal 
restricted antigen) antibodies such as anti-(fetal fibronectin) 
antibodies; or anti-(fetal restricted antigen class) antibodies such as 
anti-(fibronectin) antibodies; and the like. This invention also includes 
kits comprising one of the above insoluble support reagents, alone or in 
combination with labeled reagent antigen or antibodies. A preferred 
embodiment of a kit of this invention comprises an anti-(fetal 
fibronectin) antibody adhered to an insoluble support in combination with 
a labeled anti-(fibronectin) antibody.

DETAILED DESCRIPTION OF THE INVENTION 
The method of this invention involves the detection of uniquely fetal or 
placental restricted material in a sample expelled by a suspected 
spontaneous abortion or removed during a dilation and curettage or a 
therapeutic abortion procedure. Since the fetal restricted antigens are 
not present in significant quantities in maternal blood, the presence of 
maternal blood in the sample does not interfere with the tests. 
The term "fetal restricted antigen" as used hereinafter is defined to mean 
a uniquely fetal or placental derived material, which is either not 
present in maternal serum, plasma or urine, or is not present in 
significant amounts in maternal serum, plasma or urine. Any substance 
meeting this definition is intended to be included within the meaning of 
the term, including both antigenic materials and proteins and other 
substances which are not antigenic in their purified form but which have 
unique epitopes which can be bound by preferentially-binding antibodies. 
An example of a fetal restricted antigen is the fetal fibronectin which 
binds preferentially with the FDC-6 monoclonal antibody described by 
Matsuura, H. and Hakomori, S., Proc. Natl. Acad. Sci. USA. 82:6517-6521 
(1985). 
The term "fetal restricted antigen class" as used herein is defined to mean 
a class or group of antigens of which the "fetal restricted antigen" is a 
member. For example, fetal fibronectin is a fetal restricted antigen 
member of the total fibronectin class or group. 
The term "nonrestricted pregnancy antigen" as used herein is defined to 
mean compounds or substances which can be detected in body fluids such as 
serum, plasma, or urine to indicate pregnancy and which are present in 
significant amounts in maternal serum, plasma, or urine. 
The term "antibody" as used herein is defined to include antibodies of 
classes IgG, IgM, IgA, IgD, and IgE, and fragments and hybrid derivatives 
of antibodies including Fab, and F(ab').sub.2 fragments of antibodies. 
Antibodies may be polyclonal or monoclonal. Generally, monoclonal 
antibodies are preferred for use in the methods of this invention. 
In the method of this invention, a sample is procured which is believed to 
represent material expulsed from the uterus. Such materials can be tissues 
removed during a therapeutic abortion or a D&C (dilation and curettage). 
Alternatively, the material may be vaginal discharge which is believed to 
be indicative of a spontaneous abortion, or miscarriage. The sample 
generally comprises fluid and particulate solids, and may contain tissue 
matter, vaginal or cervical mucus, other vaginal or cervical secretions, 
cells or cell debris, amniotic fluid, or other fetal or maternal 
materials. The sample is assayed to determine the presence or quantity of 
fetal restricted antigen in the sample. The sample can be procured from 
the vaginal cavity with with a swab having a dacron or other fibrous tip, 
aspirator, suction device, lavage device or the like. The sample can 
represent tissues removed during a dilation and curettage procedure or a 
therapeutic abortion, or can be procured using a feminine protection pad 
in the case of a suspected spontaneous abortion. The sample is transferred 
to a suitable container for storage and transport to the testing 
laboratory. 
It is important that the sample be dispersed in a liquid which preserves 
the sensitive protein analytes which are unstable in the sampled 
composition. The storage and transfer medium should prevent decline in the 
protein analyte level during storage and transport. A suitable preserving 
solution for storage and transfer is described in U.S. patent application 
Ser. No. 244,969, filed Sep. 15, 1988, the entire contents of which are 
hereby incorporated by reference in their entirety. 
Detection of the fetal restricted antigen can be achieved by binding the 
fetal restricted antigen in a test sample with an antibody which binds 
preferentially with an epitope of the fetal restricted antigen, and 
determining the presence or absence of this binding. Immunological methods 
are most convenient for carrying out this method because of their 
specificity. The term "immunoassays" as used herein is defined to mean any 
method using a preferential binding of a fetal restricted antigen with a 
second material, i.e., a binding partner, usually an antibody or fragment 
thereof, or another substance having an antigen binding site which binds 
preferentially with an epitope of the fetal restricted antigen. 
Preferential binding as used herein refers to binding between binding 
partners which is selective and generally specific, and which demonstrates 
less than 10%, preferably less than 5%, cross-reactive nonspecific 
binding. For example, when the analyte is fetal fibronectin, the 
anti-(fetal fibronectin) antibody is less than 10%, and preferably less 
than 5%, cross-reactive with adult fibronectins. 
Included within the scope of this invention are all immunoassay methods 
including this step, including but not limited to sandwich, competition, 
agglomeration, precipitation, transistor bridge probe, light disturbing, 
light scattering, and ultrasonic probe immunoassays, for example. 
In one sandwich embodiment of this invention, the sample is contacted with 
an insoluble support to which anti-(fetal restricted antigen) antibody is 
adhered to effect binding of fetal restricted antigen in the sample to the 
insoluble support. The insoluble support is then contacted with an 
unlabeled or labeled anti-(fetal restricted antigen) antibody, which binds 
with the fetal restricted antigen adhering to the insoluble support to 
label and measure the captured fetal restricted antigen. 
An antibody which binds with a class of substances including the analyte 
fetal restricted antigen can be substituted for the anti-(fetal restricted 
antigen) antibody capture antibody or the anti-(fetal restricted antigen) 
antibody sandwiching antibody. For example, anti-(fetal fibronectin) 
antibody can be adhered to the insoluble support, and labeled or unlabeled 
anti-(fibronectin) antibody can be used to label the captured antigen. 
Alternatively, anti-(fibronectin) antibody can be adhered to the insoluble 
support, and labeled or unlabeled anti-(fetal fibronectin) antibody is 
used to label the captured antigen. 
The sample can also be tested by a competition immunoassay procedure. The 
sample is mixed with labeled reagent antibody or antigen and incubated 
with an insoluble support to which an anti-(fetal restricted antigen) 
antibody or reagent fetal restricted antigen is adhered, competition 
occurring between the reagents for binding with the sample analyte. The 
label ultimately adhering to the insoluble support or remaining in the 
solution is then determined. 
The sample can also be tested by known dip stick, agglomeration, 
precipitation, semiconductor bridge probe, and similar procedures, 
preferably involving a preferential binding reaction between a fetal 
restricted antigen and a binding partner therefor. 
Anti-(fetal restricted antigen) antibody can be obtained from fetal 
restricted antigens, preferably from highly purified fetal restricted 
antigens, by conventional antiserum or monoclonal techniques. This 
invention will be described hereinafter with respect to the detection of 
fetal fibronectin, for purposes of clarity, and not by way of limitation: 
the detection of any fetal restricted antigen is intended to be within the 
scope of this invention. 
Fetal fibronectin is purified from amniotic fluid as described by Engvall 
and Ruoslahti, Int. J. Cancer. 20:1-5 (1977). Anti-(fetal fibronectin) 
antibody can be derived from fetal fibronectin by conventional antiserum 
techniques or by monoclonal antibody techniques. 
Polyclonal anti-(fetal restricted antigen) antibody can be obtained by 
immunizing an animal such as a rabbit, guinea pig, rat or goat with 
concentrated fetal restricted antigen, such as fetal fibronectin, removing 
serum from the immunized animal, and separating the immunoglobulins from 
the serum, for example by ammonium sulfate precipitation. The principal 
antibodies useful in the method of this invention are IgG and IgM 
antibodies, although the IgD, IgE and IgA antibodies can also be used if 
available in sufficient quantity. The fetal fibronectin antibodies are 
then affinity purified using conventional affinity chromatography 
techniques such as those described by Mishell and Shilgi in SELECTED 
METHODS IN CELLULAR IMMUNOLOGY. San Francisco: Freeman (1980), Goding, J., 
MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE. New York: Academic Press 
pp 111-114 (1983), and Parikh, I., et al, C&EN (Aug. 26, 1985), the entire 
contents of each of which are hereby incorporated by reference. Suitable 
absorbents for use in affinity chromatography include cross-linked agarose 
and cross-linked polyacrylamides to which the fetal restricted antigen 
antibody is covalently bonded. For removal of antibodies cross-reacting 
with adult fibronectins, the antibody serum is passed through columns to 
which are coupled adult fibronectins. A portion of the eluant containing 
the remaining antibody can then be passed through a fetal fibronectin 
column and eluted to yield the affinity purified antibody. 
In these procedures, the antibody solution can be applied to the column in 
a phosphate buffered saline solution, and the antibodies can be eluted 
with a 2.5M NaSCN solution, pH 8.0. Antibody concentration, if desired, 
can be achieved by negative pressure dialysis or ultrafiltration. The 
antibody solution is stable at temperature of 4.degree. C. or less. 
Repetition of the column separation procedures is continued until the 
desired separation and purity is achieved. 
Monoclonal anti-(fetal restricted antigen) antibody can be obtained by the 
methods of Galfre and Milstein, Meth. Enzym. 73:1 (1981), immunizing mice 
with fetal restricted antigens to obtain the spleen cells for 
hybridization. Suitable procedures are described by Goding, MONOCLONAL 
ANTIBODIES: PRINCIPLES AND PRACTICE. New York: Academic Press (1983) pp 
56-97, the entire contents of which are hereby incorporated by reference. 
For production of fetal fibronectin, the procedures described by Matsuura 
and Hakomori, Proc. Natl. Acad. Sci. USA. 82:6517-6521 (1985) can be 
followed, replacing the tumor fibronectin with fetal fibronectin. 
Anti-(fetal restricted antigen class) antibodies of both polyclonal and 
monoclonal varieties are generally well known and available either 
commercially or from publicly available hybridoma deposits. For example, 
anti-(fibronectin) monoclonal antibodies can be derived from clone samples 
from ATCC HB 91 (American Type Culture Collection, Rockville, Md.). Other 
such antibodies are described in Japanese Patent Application 60091264 
(DIALOG database file 351, WPI Acc. No. 85-161617/27) and U.S. Pat. No. 
4,325,867. 
Preferentially binding antibody fragments suitable for use in the kit and 
method of this invention can be made from the respective monoclonal or 
polyclonal antibodies by conventional enzyme or chemical fragmentation 
procedures. Suitable procedures are described by Tijssen, P. LABORATORY 
TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: PRACTICE AND THEORIES OF 
ENZYME IMMUNOASSAYS. New York: Elsevier (1985), for example. 
Antigen and antibody reagents can be bonded to an insoluble support by 
conventional processes. Antigen binding methods suitable for binding 
antigens to insoluble supports such as those described in U.S. Pat. Nos. 
3,234,096, 3,236,732, 3,309,275, 3,873,683, 3,991,175, 4,003,988, 
4,016,250, 4,033,723, 4,071,314, 4,348,207, and 4,419,453, for binding 
antigens to latex particles and erythrocytes, for example, can be used. 
Procedures for binding of antibodies to insoluble supports are described 
in U.S. Pat. Nos. 3,551,555, 3,553,310, 4,048,298 and RE-29,474, for 
example. Procedures for binding of antibodies to polystyrene by adsorption 
are described in U.S. Pat. Nos. 3,646,346 and 4,092,408, for example. For 
purposes of clarity and not by way of limitation, the binding procedures 
are described hereinafter with respect to binding antibodies to insoluble 
supports. These procedures are equally suitable for binding the reagent 
fetal restricted antigens to insoluble supports. 
A variety of materials can be used as the insoluble support, the primary 
consideration being the binding of the anti-(fetal restricted antigen) 
antibody or the anti-(fetal restricted antigen class) antibody to the 
surface, and the absence of interference with the reagent binding reaction 
or with other reactions which can be employed to determine the presence 
and extent of the binding reaction. Organic and inorganic polymers, both 
natural and synthetic, can be used as the insoluble support. Examples of 
suitable polymers include polyethylene, polypropylene, polybutylene, 
poly(4-methylbutylene), butyl rubber, silastic polymers, polyesters, 
polyamides, cellulose and cellulose derivatives (such as cellulose 
acetate, nitrocellulose and the like), acrylates, methacrylates, vinyl 
polymers (such as polyvinyl acetate, polyvinyl chloride, polyvinylidene 
chloride, polyvinyl fluoride, and the like), polystyrene and styrene graft 
copolymers, rayon, nylon, polyvinylbutyrate, polyformaldehyde, etc. Other 
materials which can be used as the insoluble support can the latexes of 
the above polymers, silica gel, silicon wafers, glass, paper, insoluble 
protein, metals, metalloids, metal oxides, magnetic materials, 
semi-conductive materials, cermets and the like. In addition are included 
substances which form gels, such as proteins such as gelatins, 
lipopolysaccharides, silicates, agarose, polyacrylamides or polymers which 
form several aqueous phases such as dextrans, polyalkylene glycols 
(alkylene with 2 to 3 carbon atoms) or surfactants, e.g. amphophilic 
compounds such as phospholipids, long chain (12-24 carbon atoms) alkyl 
ammonium salts and the like. 
A preferred diagnostic support of this invention comprises a nylon or 
nitrocellulose membrane. An alternate diagnostic support is made from a 
polystyrene, styrene copolymers such as styrene-acrylonitrile copolymers, 
or polyolefins such as polyethylene or polypropylene, and acrylate and 
methacrylate polymers and copolymers. The anti-(fetal restricted antigen) 
reagent antibody or the other antibody reagents can be bound to the 
insoluble support by adsorption, ionic bonding, van der Waals adsorption, 
electrostatic bonding, or other non-covalent bonding, or it can be bound 
to the insoluble support by covalent bonding. A particularly advantageous 
support for this procedure comprises a microtiter plate having a plurality 
of wells. The well surface or plastic cup inserts therein can constitute 
the antigen or antibody support. If the determination will require the use 
of fluorometric measurements, the microtiter plate or the well inserts are 
advantageously opaque to light so that excitation light applied to a well 
does not reach or influence contents of the surrounding wells. 
Procedures for non-convalent bonding are described in U.S. Pat. No. 
4,528,267. Procedures for covalently bonding antibodies and antigens to 
insoluble supports are described by I. Chibata in IMMOBILIZED ENZYMES. 
Halsted Press: New York (1978) and A. Cuatrecasas, J. Bio. Chem. 245:3059 
(1970), the entire contents of which are hereby incorporated by reference. 
The surface can be coated with a protein and coupled with the antibody or 
antigen using procedures described in U.S. Pat. No. 4,210,418 using 
glutaraldehyde as a coupling agent, for example. In an alternate 
procedure, the well can be coated with a layer having free isocyanate 
groups such as a polyether isocyanate, and application of the antibody or 
antigen in aqueous solution thereto effects the requisite bonding. In yet 
another procedure, the antibody or antigen can be coupled to a 
hydroxylated material by means of cyanogen bromide as described in U.S. 
Pat. No. 3,720,760. 
The insoluble supports are preferably "blocked" to reduce nonspecific 
binding. The choice of suitable blocking agents is determined by the type 
of insoluble support. For example, for polystyrene supports, suitable 
blocking agents include water-soluble non-immune animal proteins. Suitable 
water-soluble non-immune animal proteins include bovine (BSA), human, 
rabbit, goat, sheep, and horse serum albumins; casein and non-fat milk; 
ovalbumin, glycoproteins, and the like. 
The same blocking agents can also be used for nylon and nitrocellulose 
supports. However, a preferred blocking agent for nitrocellulose or nylon 
membrane supports is non-fat milk or casein. An optimum blocking agent for 
these membrane supports is an aqueous solution containing from 1 to 5 wt. 
% non-fat dried milk or casein, and nonionic surfactants such as 
polyoxyethylene sorbitan derivatives and polyoxyethylene ethers. 
The labeled reagent fetal restricted antigen, anti-(fetal restricted 
antigen) antibody, anti-(fetal restricted antigen class) antibody and 
anti-(sandwiching antibody) reagent antibodies of this invention can be 
prepared by conventional procedures for attaching labels to proteins, 
preferably with suitable protection of antibody binding sites. The labels 
can be bonded or coupled to the protein reagents by chemical or physical 
bonding. Ligands and groups which can be conjugated to the reagent antigen 
or antibodies of this invention include elements, compounds or biological 
materials which have physical or chemical characteristics which can be 
used to distinguish the reagents to which they are bonded from compounds 
and materials in the sample being tested. 
Labeling procedures are described hereinafter with respect to labeling 
antibodies for purposes of clarity and not by way of limitation, and the 
procedures described are generally suitable for labeling any proteinaceous 
compound or substance, such as the reagent fetal restricted antigens 
herein. 
Radiolabeled anti-(fetal restricted antigen) antibodies of this invention 
can be used for in vitro diagnostic tests. The specific activity of a 
tagged antibody depends upon the half-life, isotopic purity of the 
radioactive label and how the label is incorporated into the antigen or 
antibody. Table A lists several commonly used isotopes, their specific 
activities and half-lives. In immunoassay tests, the higher the specific 
activity, in general, the better the sensitivity. 
TABLE A 
______________________________________ 
Specific Activity of Pure 
Isotope Isotope (Curies/mole) 
Half-Life 
______________________________________ 
14.sub.C 6.25 .times. 10.sup.1 
5720 years 
3.sub.H 2.91 .times. 10.sup.4 
12.5 years 
35.sub.S 1.50 .times. 10.sup.6 
87 days 
125.sub.I 2.18 .times. 10.sup.6 
60 days 
32.sub.P 3.16 .times. 10.sup.6 
14.3 days 
131.sub.I 1.62 .times. 10.sup.7 
8.1 days 
______________________________________ 
Procedures for labeling antibodies with radioactive isotopes listed in 
Table A are generally known in the art. Tritium labeling procedures are 
described in U.S. Pat. No. 4,302,438, for example. Iodinating, tritium 
labeling and .sup.35 S labeling procedures especially adapted for 
antibodies are described by Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND 
PRACTICE. New York: Academic Press (1983) pp 124-126 and the references 
cited therein. Other procedures for iodinating antibodies are described by 
Hunter and Greenwood, Nature. 144:945 (1962) and David et al, Biochem. 
13:1014-1021 (1974) and in U.S. Pat. Nos. 3,867,517 and 4,376,110. 
Examples of suitable systems, coupling procedures and substrate reactions 
therewith are disclosed in U.S. Pat. Nos. RE-31,006, 3,654,090, 4,214,048, 
4,289,747, 4,302,438, 4,312,943, 4,376,110 and the references cited 
therein, for example. Examples of other suitable systems are described by 
Pesce et al, Clin. Chem. 20:353-359 (1974) and Wisdom, G., Clin. Chem. 
22:1243 (1976). 
A list of suitable enzyme classes which can be used for labeling and 
specific examples for each class follow: 
TABLE B 
______________________________________ 
Class Enzyme Example 
______________________________________ 
Hydrolases Carbohydrases 
Amylases 
Nucleases Polynucleotidase 
Amidases Arginase 
Purine deaminases Adenase 
Peptidases Aminopolypeptidase 
Proteinases Pepsin 
Esterases Lipases 
Iron Enzymes Catalase 
Copper Enzymes Tyrosinases 
Enzymes containing Coenzymes 
Alcohol dehydrogenase 
Enzymes reducing cytochrome 
Succinic dehydrogenase 
Yellow enzymes Diaphorase 
Mutases Glyoxalase 
Demolases Aldolase 
Oxidases Glucose oxidase 
Horseradish peroxidase 
______________________________________ 
A list of suitable enzymes are described in Hawk, et al. PRACTICAL 
PHYSIOLOGICAL CHEMISTRY, New York: McGraw-Hill pp 306-397 (1954). 
Fluorogenic and chromogenic enzymes (enzymes in the presence of which a 
selected substrate will produce a fluorescent or chromogenic product) are 
useful labeling moieties. Methods for selectively conjugating enzymes to 
antibodies without impairing the ability of the antibody to bind with 
antigen and for conjugating enzymes to proteinaceous reagents are well 
known in the art. 
Suitable enzymes and procedures for coupling them to antibodies are 
described by I. Chibata in IMMOBILIZED ENZYMES. Halsted Press: New York 
(1978); A. Cuatrecasas, J. Bio. Chem. 245:3059 (1970); Wilson, M. et al, 
INTERNATIONAL CONFERENCE IN IMMUNOFLUORESCENCE AND RELATED STAINING 
TECHNIQUES. W. Knapp et al, editors. Amsterdam: Elsevier pp 215-244 
(1978); Sullivan, M. et al, Ann. Clin. Biochem. 16:221-240 (1979); Nygren, 
H. et al, Med. Biol. 57:187-191 (1979); Gadkari, D. et al, J. Virol. Meth. 
10:215-224 (1985); Tijssen, P. et al, Anal. Biochem. 136:451-457 (1984); 
Tsuruta, J. et al, J. Histochem. Cytochem. 33:767-777 (1985); Ishikawa, 
E., J .Immunoassay. 4:209-327 (1983); and in U.S. Pat. No. 4,190,496, for 
example, the entire contents of the above listed references being hereby 
incorporated by reference in their entireties. 
The preferred enzymes and suitable substrates corresponding thereto include 
horseradish peroxidase for which suitable substrates are 
o-phenylenediamine, m-phenylenediamine, o-dianisidine, and 
4-chloro-.alpha.-napthol. They also include .beta.-galactosidase for which 
suitable substrates are 4-methylumbelliferyl-.beta.-D-galactoside, 
p-nitrophenyl-.beta.-D-galactose, p-nitrophenol, 
o-nitrophenyl-.beta.-D-galactose, and o-nitrophenol, for example. They 
include alkaline phosphatase for which suitable substrates are 
p-nitrophenylphosphate, indoxyl phosphate, and 5-bromo-3-chloroindoxyl 
phosphate, for example. 
Examples of suitable procedures for enzyme labeling the antibody include 
the use of carbodiimides, dialdehydes, and bifunctional coupling reagents. 
Linkage of enzymes through amide groups can be achieved by treating the 
proteins with thionyl chloride, N-hydroxysuccinimide or similar reagents 
in an anhydrous solvent such as dimethylformamide, dioxane, 
dimethylsulfoxide, tetrahydrofuran, or the like. Alternative coupling 
agents include carbodiimides such as 
1-ethyl-3-(3-(N,N'-dimethylamino)propyl)-carbodiimide, 
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide methyl-p-toluenesulfonate, 
succinimidyl 4-(N-maleimidoethyl)-cyclohexane-1-carboxylate, and 
succinimidyl 3-(2-pyridyldithio)-propionate, for example. 
The carbohydrate moiety of an enzyme can also be oxidized to an aldehyde 
and reacted with lysyl amino groups of immunoglobulins to form a Schiffs 
base. Reduction with sodium borohydride effects a stable linkage of enzyme 
and antibody. Horseradish peroxidase with antibody can be efficiently 
linked to immunoglobulins by the method of Wilson et al, INTERNATIONAL 
CONFERENCE IN IMMUNOFLUORESCENCE AND RELATED STAINING TECHNIQUES. W. Knapp 
et al, editors. Amsterdam: Elsevier pp 215-244 (1978). 
Fluorophore and chromophore labeled antibodies can be prepared from 
standard fluorescent moieties known in the art. Since antibodies and other 
proteins absorb light having wavelengths up to about 310 nm, the 
fluorescent moieties should be selected to have substantial absorption at 
wavelengths above 310 nm and preferably above 400 nm. A variety of 
suitable fluorescers and chromophores are described by Stryer, Science. 
162:526 (1968) and Brand, L. et al, Ann. Rev. Biochem. 41:843-868 (1972). 
The antibodies can be labeled with fluorescent chromophore groups by 
conventional procedures such as those disclosed in U.S. Pat. Nos. 
3,940,475, 4,289,747 and 4,376,110, for example. 
One group of fluorescers having a number of the desirable properties 
described above are the xanthene dyes, which include the fluoresceins 
derived from 3,6-dihydroxy-9-phenylxanthhydrol and resamines and 
rhodamines derived from 3,6-diamino-9-phenylxanthydrol and lissanime 
rhodamine B. The rhodamine and fluorescein derivatives of 
9-o-carboxyphenylxanthhydrol have a 9-o-carboxyphenyl group. Fluorescein 
compounds having reactive coupling groups such as amino and isothiocyanate 
groups such as fluorescein isothiocyanate and fluorescamine are readily 
available. 
Another group of fluorescent compounds are the naphthylamines, having an 
amino group in the alpha or beta position. Included among the 
naphthylamino compounds are 1-dimethylaminonaphthyl-5-sulfonate. 
1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalene 
sulfonate. Other dyes include 3-phenyl-7-isocyanatocoumarin; acridines 
such as 9-isothiocyanatoacridine and acridine orange; 
N-[p-(2-benzoxazolyl)phenyl]maleimide; benzoxadiozoles such as 
4-chloro-7-nitrobenzo-2-oxa-1,3-diazole and 
7-(p-methoxybenzylamino)-4-nitrobenzo-2-oxa-1,3-diazole; stilbenes such as 
4-dimethylamino-4'-isothiocyanatostilbene and 
4-dimethylamino-4'-maleimidostilbene; 
N,N'-dioctadecycloxacarboxyamine-p-toluenesulfonate; pyrenes such as 
8-hydroxy-1,3,6-pyrenetrisulfonic acid, 1-pyrenebutyric acid, merocyanine 
540, rose bengal, 2,4-diphenyl-3(2H)-furanone, o-phthaldehyde, as well as 
other readily available fluorescing molecules. These dyes either have 
active functionalities or such functionalities can be readily introduced. 
Antibodies can be labeled with fluorochromes or chromophores by the 
procedures described by Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND 
PRACTICE. New York: Academic Press (1983) pp 208-249. The concentration of 
fluorochrome is selected according to the table of Goding, p 229. For 
example, fluorescein isocyanate (1.0 mg/mL) or rhodamine isocyanate (10.0 
mg/mL) in DMSO is prepared, and the desired volume (1-10% of total protein 
solution volume) is added to the protein solution dropwise, with stirring. 
The reaction proceeds for two hours, shielded from light. The product is 
purified by gel filtration on SEPHADEX G-25 gel in PBS containing 0.1% 
NaNO.sub.3 to separate the unreacted or hydrolyzed fluorochrome. The 
absorbence of the conjugate is measured at 280 nm and at its peak in the 
visible region (495 nm for fluoresceinated antibody and 550 nm for 
rhodaminated antibody). The fluorochrome to protein ratio is calculated 
according to the procedure of Goding, MONOCLONAL ANTIBODIES: PRINCIPLES 
AND PRACTICE. New York: Academic Press (1983) pp 224-225. Conjugates are 
stored at 4.degree. C. protected from light until use. If the antibody 
solution concentration is less than 1 mg/mL, BSA is added to the solution 
to a final concentration of 1 mg/mL. 
The antibodies and reagent antigen used in the methods of this invention 
can be covalently bonded to avidin or biotin in one embodiment of this 
invention. Suitable binding procedures involve cross-linking through a 
bifunctional cross-linking agent. Suitable bifunctional compounds are 
described by Peters, K. et al, Ann. Rev. Biochim. 46:523 (1977). Alkyl 
imidates show a high degree of specificity among the functional groups 
presented to them by a protein. The reaction is specific for primary amino 
groups. Examples of suitable coupling reagents include amidoesters such as 
dimethylmalonimidate, azides such as the acyl azide of tartryl diazide 
which reacts readily with immuno groups to produce amide linkages. Aryl 
dihalides (e.g., 1,5-difluoro-2,4-dinitrobenzene, or 4,4'-difluoro- 
3,3'-dinitrophenyl sulfone, glutaraldehyde, 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, dimaleinide, 
mixed anhydride, m-maleamidobenzoyl N-hydroxysucciinimide ester, and other 
known cross-linking agents. 
The foregoing reagents provide essentially irreversible bonds. Bifunctional 
agents with functional groups such as disulfide or glycol may be used. 
These provide bonds which can be broken after the cross-linking reaction, 
if desired. Such reagents include dimethyl 3,3'-dithiobispropionimidate, 
succinimidylpropionimidate, N-(3-fluoro-4,6-dinitrophenyl)-cystamine, 
tartryl diazide, tartryl di(glycylazide) and tartryl di(epsilon-amino 
caproylazide). 
In other instances, the bonds can be formed directly between the reagents 
themselves. For example, antibody can be bound to biotin through 
functional groups on the respective materials. As a specific example, 
biotin can be treated with periodate and reacted with antibody to give a 
Schiff base formation without inhibiting the biotin to avidin binding or 
blocking immunological activity of the antibody. Avidin-conjugated and 
biotinylated reagents are available from Vector Laboratories, Burlingame, 
Calif. 
Known techniques using bifunctional cross-linking agents include the 
following: (a) a one-step glutaraldehyde linkage, Avrameas, S., 
Immunochem. 6:43 (1969); (b) two-step glutaraldehyde linkage, Avrameas, 
S., Immunochem. 8:1175 (1971); and (c) dimaleimide linkage, Kato, K. et 
al, Euro. J. Biochem. 62:285 (1966). 
Antibodies can be labeled with metallic radionuclides according the 
procedure of Hnatowich, D. et al. J. Appl. Rad. 35:554-557 (1984) and 
Buckley, R et al. Fed. Eur. Biochem. Soc. 166:202-204 (January 1984). In 
this procedure the antibodies are conjugated with a chelating agent such 
as diethylenetriaminepentaacetic acid which is capable of forming a 
chelate with the metallic radionuclide. A suspension of 0.1 mg/mL of the 
bicyclic anhydride of DTPA (diethylenetriaminepentaacetic acid) is 
prepared in a dry solvent such as chloroform, ether or dry DMSO. An 
aliquot is removed to a clean, dry tube sufficient to provide a DTPA to 
immunoglobulin molar ratio of 1:1 and evaporated under nitrogen. A 10-20 
microliter portion of the antibody solution used (10-20 mg/mL) in 0.05M 
bicarbonate buffer in saline, pH 7.0-7.5 is added to the dry DTPA, and the 
contents are agitated for 0.5-1.0 minute. The coupled protein preparation 
is diluted to 0.2 mL with the same buffer solution and purified on a 5 cm 
gel filtration column with SEPHADEX G-50 gel, using a saline eluant. The 
coupling efficiency is determined before purification by the addition of 
"chelation-grade" .sup.111 In in 0.5M acetate buffer solution, pH 6.0. 
Thin layer chromatography is used to separate the DTPA coupled antibody 
for calculation of the coupling efficiency. The DTPA-coupled antibodies 
can be stored at 4.degree. C. until needed for binding with metallic 
radionuclides such as .sup.111 In+3, .sup.212 Bi+3 and .sup.68 Ga+3, for 
example. 
In one sandwich embodiment of the immunoassay methods of this invention, an 
insoluble support to which anti-(fetal restricted antigen) antibody is 
adhered is contacted with a test sample diluted with an aqueous buffer 
solution such as phosphate buffer solution (PBS), pH 6 to 8 and preferably 
from 7.2 to 7.6 for a sufficient time to permit binding of fetal 
restricted antigen in the test sample with the anti-(fetal restricted 
antigen) antibody on the insoluble support, and then removing the sample 
from the support. The incubation time should be sufficient to permit 
substantial binding to occur, the time being temperature dependent. 
Suitable incubation times are from 30 to 240 minutes at temperatures 
within the range of from 16.degree. to 40.degree. C., the preferred 
contact time being at least 60 minutes at temperatures within the range of 
from 20.degree. to 26.degree. C. 
Residual sample solution is then removed from the support by use of a rinse 
solution. Any conventional rinse solution can be used. A suitable rinse 
solution is described in U.S. Pat. No. 4,528,267. It is an aqueous 
phosphate buffer solution having a phosphate molarity of from 0.0001 to 
0.05, a pH of from 6 to 8 and containing from 0.001 to 0.1 weight percent 
of nonionic surfactant. Suitable nonionic surfactants include 
polyoxyethylene ethers (BRIJ such as lauryl, cetyl, oleyl, stearyl, and 
tridecyl polyoxyethylene ethers; polyoxyethylene sorbitans (TWEEN such as 
polyoxyethylene sorbital monolaurate, monopalmitate, monostearate, 
monoleate, and trioleates); and other polyoxyethylene ethers (TRITON, for 
example). A preferred nonionic surfactant is octylphenoxypolyethoxy 
ethanol having 40 ethylene oxide units (TRITON X-405, Rohm and Hass 
Company). 
The insoluble support is then contacted with an antibody which will bind 
with the captured fetal restricted antigen on the insoluble support, i.e., 
the sandwiching antibody. The sandwiching antibody can be an anti-(fetal 
restricted antigen) antibody, or can be an anti-(fetal restricted antigen 
class) antibody. The sandwiching antibody can be labeled or unlabeled. In 
the event that an unlabeled sandwiching antibody is used, a tertiary 
antibody which binds with the sandwiching antibody and which bears a 
physically detectable label can be used in a conventional manner to 
determine the sandwiching antibody. 
A variety of labels have been described above. For purposes of clarity and 
not by way of limitation, the subsequent steps of the process will be 
described for anti-(fetal restricted antigen) antibodies which have been 
labeled with an enzyme, preferably a fluorogenic or chromogenic enzyme. 
The term "fluorogenic enzyme" is defined herein to refer to an enzyme 
which will produce a fluorophore product with a suitable substrate. The 
term "chromogenic enzyme" is defined herein to refer to an enzyme which 
will produce a chromophore product with a suitable substrate. 
The sandwiching antibody is applied to the insoluble support in an aqueous 
solution. The solution preferably contains suitable salts and buffers to 
preserve the reactants and facilitate the binding reaction. For example, 
the solution can contain bovine serum albumin (BSA), phosphate buffer 
solution (PBS), and a mild surfactant such as polyoxyethylene sorbitan 
ester employed in the above-described rinse solution. The incubation is 
continued for sufficient time to permit the sandwiching antibody to bind 
with exposed fetal restricted antigen epitopes, if any, adhering to the 
insoluble support. The preferred incubation times and temperatures are as 
set forth above for the binding of insolubilized reagent anti-(fetal 
restricted antigen) antibody with the sample fetal restricted antigen. 
The sandwiching antibody solution is then removed from the insoluble 
support, and the support is rinsed with a rinse solution such as described 
above, to remove any residual, unbound material. 
If the sandwiching antibody is unlabeled, an enzyme labeled antibody or 
other binding agent which binds preferentially with the sandwiching 
antibody is applied to the insoluble support in an aqueous solution. The 
solution preferably contains suitable salts and buffers to preserve the 
reactants and facilitate the binding reaction. For example, the solution 
can contain bovine serum albumin (BSA), phosphate buffer solution (PBS), 
and a mild surfactant such as polyoxyethylene sorbitan ester employed in 
the above-described rinse solution. The incubation is continued for 
sufficient time to permit labeled anti-(sandwiching antibody) antibody to 
bind with exposed sandwiching antibody epitopes, if any, adhering to the 
insoluble support. The preferred incubation times and temperatures are as 
set forth for the binding of insolubilized reagent anti-(fetal restricted 
antigen) antibody with the sample fetal restricted antigen. 
The labeled antibody solution is then removed from the insoluble support, 
and the support is rinsed with a rinse solution such as described above, 
to remove any residual, unbound labeled material. 
In a next step, the insoluble support is contacted with an aqueous solution 
of a substrate which undergoes a reaction in the presence of the enzyme to 
release fluorescent or chromogen compound into the solution. Suitable 
substrates and the enzymes which which they can be converted are described 
in U.S. Pat. Nos. 4,190,496 and 4,528,267, for example. The support is 
contacted with an aqueous solution of the substrate containing from 
10.sup.-2 to 10.sup.-10 molar concentrations of the substrate. Substrate 
molar concentrations of from 10.sup.-4 to 10.sup.-5 are preferred. 
Preferred additional reagents and buffers in the substrate solution 
include 2-amino-2-methyl-1-propanol buffer and magnesium chloride, for 
example. 
The substrate solution is incubated with the insoluble support for 
sufficient time for the reaction yielding the fluorophore or chromophore 
to occur. At temperatures of from 18.degree. to 40.degree. C., incubation 
times of from 5 to 240 minutes can be used. Preferably, the temperature is 
within the range of from 20.degree. to 26.degree. C., and the incubation 
time is from 30 to 90 minutes. 
The fluorescent or chromophore level in the solution is then measured. The 
equipment and procedures for determining the level of fluorescence or 
chromophore level in the substrate solutions are those conventionally used 
in the art. The level of fluorescence or chromogen in solution is a 
function of the enzyme concentration on the insoluble support which is, in 
turn, a function of the amount of fetal restricted antigen in the sample. 
The concentration of the fetal restricted antigen can be determined by 
comparing the fluorescence or chromophore level of the solution with 
respective fluorescence or chromophore levels obtained with control 
solutions containing known concentrations of fetal restricted antigen. 
The sandwich procedure can be modified to use a fetal restricted antigen 
class binding antibody as either the capture or sandwiching antibody. In 
these embodiments, an anti-(fetal restricted antigen class) antibody, such 
as an anti-(fibronectin) antibody, is adhered to the insoluble support, 
and a labeled or unlabeled anti-(fetal restricted antigen) antibody is 
applied as the sandwiching antibody. Alternatively, the anti-(fetal 
restricted antigen) antibody can be adhered to the insoluble support, and 
a labeled or unlabeled anti-(fetal restricted antigen class) antibody is 
used to sandwich the captured antigen. 
In one membrane embodiment of the immunoassay methods of this invention, an 
insoluble support to which anti-(fetal restricted antigen) antibody is 
adhered is contacted with a test sample diluted with an aqueous buffer 
solution such as phosphate buffer solution (PBS), pH 6 to 8 and preferably 
from 7.2 to 7.6 for a sufficient time to permit binding of fetal 
restricted antigen in the sample with the anti-(fetal restricted antigen) 
antibody on the insoluble support. The time required for binding is very 
small in a flow through system. Suitable incubation times can be one 
second up to 20 minutes at temperatures within the range of from 
16.degree. to 40.degree. C., the preferred contact time being less than 
one minute and optimally from 10 seconds to 2 minutes. 
The insoluble support is then contacted with an antibody which will bind 
with the captured fetal restricted antigen on the insoluble support, i.e., 
the sandwiching antibody. The sandwiching antibody can be labeled or 
unlabeled. In the event that an unlabeled sandwiching antibody is used, a 
secondary antibody which binds with the sandwiching antibody and which 
bears a physically detectable label can be used in a conventional manner 
to determine the sandwiching antibody. 
A variety of labels have been described above. For purposes of clarity and 
not by way of limitation, the subsequent steps of the process will be 
described for anti-(fetal restricted antigen) antibodies which have been 
labeled with an enzyme, preferably a fluorogenic or chromogenic enzyme. 
The sandwiching antibody is applied to the insoluble support in an aqueous 
solution. The solution preferably contains suitable salts and buffers to 
preserve the reactants and facilitate the binding reaction. For example, 
the solution can contain bovine serum albumin (BSA), phosphate buffer 
solution (PBS), and a mild surfactant such as polyoxyethylene sorbitan 
ester employed in the above-described rinse solution. The incubation is 
continued for sufficient time to permit the sandwiching antibody to bind 
with exposed fetal restricted antigen epitopes, if any, adhering to the 
insoluble support. The preferred incubation times and temperatures are as 
set forth for the binding of insolubilized reagent anti-(fetal restricted 
antigen) antibody with the sample fetal restricted antigen. 
The sandwiching antibody solution optionally can be removed from the 
insoluble support, and the support is rinsed with a rinse solution such as 
described above, to remove any residual, unbound labeled material. 
If the sandwiching antibody is unlabeled, an enzyme labeled antibody or 
other binding agent which binds preferentially with the sandwiching 
antibody is applied to the insoluble support in an aqueous solution. The 
solution preferably contains suitable salts and buffers to preserve the 
reactants and facilitate the binding reaction. For example, the solution 
can contain bovine serum albumin (BSA), phosphate buffer solution (PBS), 
and a mild surfactant such as polyoxyethylene sorbitan ester employed in 
the above-described rinse solution. The incubation is continued for 
sufficient time to permit labeled anti-(sandwiching antibody) antibody to 
bind with sandwiching antibody epitopes, if any, adhering to the insoluble 
support. The preferred incubation times and temperatures are as set forth 
for the binding of insolubilized reagent anti-(fetal restricted antigen) 
antibody with the sample fetal restricted antigen. 
The labeled antibody solution is then removed from the insoluble support, 
and the support is rinsed with a rinse solution such as described above, 
to remove any residual, unbound labeled material. 
In a next step of the membrane sandwich process of this invention, the 
insoluble support is contacted with an aqueous solution of a substrate 
which undergoes a reaction in the presence of the enzyme to release 
fluorogen or chromogen compound into the solution. Suitable substrates and 
the enzymes which which they can be converted, as well as additional 
components and buffers have been described above. 
The substrate solution is incubated with the insoluble support for 
sufficient time for the reaction yielding the fluorophore or chromophore 
to occur. At temperatures of from 18.degree. to 40.degree. C., incubation 
times of from 1 to 20 minutes can be used. Preferably, the temperature is 
within the range of from 20.degree. to 26.degree. C., and the incubation 
time is from 2 to 5 minutes. The fluorescence or chromogen level on the 
membrane can be measured using a reflectometer or densitometer. 
Competition embodiments of this invention using labeled reagent fetal 
restricted antigen comprise contacting a mixture of the sample and the 
labeled reagent fetal restricted antigen with an anti-(fetal restricted 
antigen) antibody adhered to an insoluble support, and determining the 
amount of label which either binds with the insoluble support or remains 
in the solution phase. 
The competition embodiments of this invention using labeled anti-(fetal 
restricted antigen) antibodies can be of more than one form. One 
embodiment using anti-(fetal restricted antigen) antibody bound to the 
insoluble support comprises contacting a mixture of the sample and labeled 
anti-(fetal restricted antigen) antibodies with an anti-(fetal restricted 
antigen) antibody adhered to an insoluble support, and determining the 
amount of label which either binds with the insoluble support or remains 
in the solution phase. Another embodiment using reagent fetal restricted 
antigen bound to the insoluble support comprises contacting a mixture of 
the sample and labeled anti-(fetal restricted antigen) antibodies with a 
fetal restricted antigen adhered to an insoluble support, and determining 
the amount of label which either binds with the insoluble support or 
remains in the solution phase. 
In each of these methods, the sample is diluted with buffer solution, 
incubated and the label determined as described above with respect to the 
sandwich immunoassay embodiments. The concentration of the limiting 
reagent is selected to permit competitive binding between the reagents, 
with the amount of label remaining on the insoluble support or in the 
solution being a variable which is a function of the amount of the analyte 
in the sample. These methods are generally well known and how to vary them 
to optimize a procedure are fully within the knowledge of a person skilled 
in the immunoassay art. 
The binding of the anti-(fetal restricted antigen) antibody and the fetal 
restricted antigen in the sample can also be determined by agglomeration 
of particles to which the anti-(fetal restricted antigen) antibody is 
adhered by fetal restricted antigen in the sample, precipitation of 
antibodies due to antibody-antigen reactions, or observations of physical 
or electrical changes which occur upon the antibody-antigen binding, using 
semiconductor bridge probes, light disturbing patterns such as are 
described in U.S. Pat. No. 4,647,544, and the like. 
If fetal restricted antigen level determinations in a test sample from a 
pregnant patient are negative, the continuation of pregnancy is indicated. 
If fetal restricted antigen level determinations in a test sample from a 
pregnant patient are positive, the termination of pregnancy is indicated. 
When a pregnant patient has had a dilation and curettage, or a therapeutic 
abortion, and there is a negative indication of fetal restricted antigen 
in the test sample, continued pregnancy, potentially an ectopic pregnancy, 
is indicated. 
Determination of the presence of fetal restricted antigen in a test sample 
from a pregnant patient indicate that pregnancy has been terminated. 
A wide variety of methods are known to a person skilled in the art for 
determining the levels of nonrestricted pregnancy indicating antigens in 
the blood or urine of a patient. Any reliable method can be used. 
Procedures for measuring hCG in plasma, serum and/or urine are described 
in U.S. Pat. Nos. 3,171,783, 3,234,096, 3,236,732, 3,298,787, 3,309,275, 
3,485,751, 3,655,838, 3,689,633, 3,862,302, 3,873,682, 3,873,683, 
3,833,304, 3,991,175, 4,003,988, 4,014,653, 4,016,250, 4,033,723, 
4,071,314, 4,094,963, 4,123,224, 4,123,509, 4,138,214, 4,208,187, 
4,210,723, 4,234,561, 4,256,629, 4,268,435, 4,270,923, 4,310,455, 
4,313,871, 4,320,111, 4,348,207, 4,371,515, 4,419,453, 4,421,896, 
4,493,793, 4,508,829, and 4,665,034, for example. Pregnancy detection by 
measuring progesterone metabolites in milk, serum or plasma (Hungary 
Patent No. T37028, WPI No. 86-023344/04); human placental lactogen in 
serum or plasma (U.S. Pat. No. 3,892,841, 4,371,515, and 4,493,793); 
estrogen steroids in urine (U.S. Pat. No. 3,955,928); luteinizing hormone 
(LH), prolactin (PRL) and/or hCG-like substances in serum, plasma or urine 
(U.S. Pat. Nos. 4,016,250, 4,094,963, and 4,320,111); pregnancy specific 
.beta..sub.1 -glycoprotein (U.S. Pat. Nos. 4,065,445 and 4,191,533); LH 
(U.S. Pat. Nos. 4,138,214 and 4,208,187); bovine pregnancy antigen in 
bovine serum or urine (European Patent Application 188,551, WPI No. 
86-042108/06); a new placental protein (U.S. Pat. No. 4,592,863); and 
early pregnancy factor WO 8605498 (WPI No. 86-264940/40) have been 
described. Methods have been described for determining pregnancy by adding 
dyes to urine (U.S. Pat. No. 2,587,221 and 3,226,196, 
dinitrophenylhydrazine; U.S. Pat. No. 3,595,620, bromocresol purple or 
chlorophenol red), by an iodine-paper test (U.S. Pat. No. 3,248,173), by 
adding other precipitating agents (U.S. Pat. No. 3,278,270), by a 
treatment of female blood with a mixture of acids and sodium chloride 
(U.S. Pat. No. 3,883,304). Pregnancy may be determined using an assay 
following the teachings of U.S. application Ser. No. 121,902 filed Nov. 
17, 1987, the contents of which are hereby incorporated by reference in 
their entireties. Any one of the above methods can be used, but methods 
such as hCG measurements are preferred. 
Kits for determining the presence of products of conception included within 
the scope of this invention generally include an anti-(fetal restricted 
antigen) antibody or reagent fetal restricted antigen adhered to an 
insoluble support; a combination of anti-(fetal restricted antigen) 
antibody adhered to an insoluble support, and either a labeled anti-(fetal 
restricted antigen) antibody, a labeled anti-(fetal restricted antigen 
class) antibody, or a labeled reagent fetal restricted antigen; a 
combination of anti-(fetal restricted antigen class) antibody adhered to 
an insoluble support together with a labeled anti-(fetal restricted 
antigen) antibody; and/or a reagent fetal restricted antigen adhered to an 
insoluble support and a labeled anti-(fetal restricted antigen) antibody. 
The kits of this invention can further comprise combinations of buffers 
for transport and storage; vials, foil packages or other containers of 
reagents of this invention; and combinations thereof. Each of the 
insoluble support structures in a foil package, vial or other packaging 
can be combined with other reagents in vials or other packages. They can 
also be combined with other, optional reagents such as enzyme substrate 
reagents in separate vials or other packages, and mechanical or optical 
devices to determine the presence and extent of antibody-antigen binding. 
This invention is further illustrated by the following specific, but 
non-limiting examples. Temperatures are given in degrees Centigrade and 
percents as weight percents unless otherwise specified. Examples which are 
constructively reduced to practice herein are presented in the present 
tense, and examples representing laboratory experiments previously reduced 
to practice are presented in the past tense. 
EXAMPLE 1 
Polyclonal Anti-(fetal fibronectin) Antibodies 
Fetal fibronectin is purified from amniotic fluid as described by Engvall 
and Ruoslahti, Int. J. Cancer. 20:1-5 (1977). 
The anti (fetal fibronectin) antibodies are elicited in rabbits using the 
immunization techniques and schedules described in the literature, e.g., 
Stollar, Meth. Enzym. 70:70 (1980), immunizing the rabbits with the fetal 
fibronectin antigen. The antiserum is screened in a solid phase assay 
similar to that used for monoclonal antibodies, e.g.., as described by 
Lange et al, Clin. Exp. Immunol. 25:191 (1976) and Pisetsky et al, J. 
Immun. Meth. 41:187 (1981). 
The IgG fraction of the antisera is purified further by affinity 
chromatography using CNBr-Sepharose 4B (Pharmacia Fine Chemicals) to which 
has been coupled fetal fibronectin. The method used for coupling is that 
recommended by the gel manufacturer, AFFINITY CHROMATOGRAPHY. Pharmacia 
Fine Chemicals, pp 15-18. 
The column is equilibrated with from 2 to 3 volumes of buffer (0.01M PBS, 
pH 7.2), and the anti-(fetal fibronectin) antibody containing solution is 
then applied to the column. The absorbency of the eluate is monitored at 
280 nm until protein no longer passes from the column. The column is then 
washed with 0.1M glycine buffer, pH 2.5, to desorb the immunoaffinity 
bound anti-(fetal fibronectin) antibody. Peak protein fractions are 
collected, pooled and dialyzed against 0.01M PBS, pH 7.2, for 24-36 hours 
at 4.degree. C. with multiple buffer changes. 
If a higher purity is desired, the affinity purified IgG can be passed 
through an adult plasma fibronectin bound affinity column by the procedure 
described above to remove any antibodies which would cross-react with 
adult plasma fibronectins. 
EXAMPLE 2 
Monoclonal Anti-(fetal antigen) Antibody 
Using purified fetal fibronectin obtained by the procedure of Example 1, 
mouse monoclonal antibodies to the fetal fibronectin are obtained using 
standard procedures of Galfre and Milstein, Meth. Enzym. 73:1 (1981) and 
Matsuura and Hakomori, Proc. Natl. Acad. Sci. USA 82:6517-6521 (1985), 
using fetal fibronectin as the antigen for immunizing the mice. The 
monoclonal antibodies are screened using a modification of the techniques 
described in the literature, e.g., Lange et al, Clin. Exp. Immunol. 25:191 
(1976) and Pisetsky et al, J. Immun. Meth. 41:187 (1981). 
Mouse monoclonal antibody is purified from ascites fluid or from hybridoma 
culture supernatants using Protein-A coupled Sepharose-4B (Pharmacia Fine 
Chemicals) according to the procedure of Tijsson, PRACTICE AND THEORY OF 
ENZYME IMMUNOASSAYS. Elsevier Science Publishers, pp 105-107 (1985). 
EXAMPLE 3 
Polyclonal Antibody Coated Microtiter Plate 
Rabbit anti-(fetal fibronectin) prepared and further purified to remove 
adult fibronectin cross-reactivity as described in Example 1 is diluted to 
10 .mu.g/mL in 0.05M carbonate buffer, pH 9.6. 100 .mu.L is dispersed into 
each well of of an IMMULON II microtiter plate (Dynatech). The plate is 
covered and incubated 4 hr at room temperature or 4.degree. C. overnight. 
The plate is washed 4 times with Wash Buffer (0.02M Tris HCl, 0.015M NaCl, 
0.05% TWEEN-20), filling and emptying the wells completely with each use. 
The plate is then blocked by dispersing into each well 200 .mu.L of a 
blocking solution (0.01M PBS, 1% BSA, 0.02% NaN.sub.3, pH 7.4) and 
incubating for 1 hour at room temperature. The wells are washed 4 times 
with Wash Buffer, as described above. The plate is now ready for 
immunoassay of samples. 
EXAMPLE 4 
Monoclonal Antibody Coated Microtiter Plate 
Goat F(ab').sub.2 anti-(mouse IgG) antibody (Tago) is diluted to 10 
.mu.g/mL in 0.05M carbonate buffer, pH 9.6. 100 .mu.L is dispersed into 
each well of an IMMULON II microtiter plate (Dynatech). The plate is 
covered and incubated 4 hours at room temperature or 4.degree. C. 
overnight. The plate is washed 4 times with Wash Buffer as described in 
Example 3. The plate is then blocked by dispensing into each well the 
Blocking Solution as described in Example 3. Mouse monoclonal anti-(fetal 
fibronectin) ascites prepared as in Example 2 is diluted 1/5000 with 0.01M 
PBS-1% BSA, pH 7.4. 100 .mu.L of the solution is dispensed into each well 
of the blocked microtiter plate. The wells are incubated, covered, for 2 
hours at room temperature or overnight at 4.degree. C. The plate is washed 
4 times with Wash Buffer as described above, and is then ready for 
immunoassay of samples. 
EXAMPLE 5 
Enzyme Labeled Antibody 
Anti-(fibronectin) antibody prepared in accordance with the procedures of 
Example 1 or Example 2 is conjugated with alkaline phosphatase following 
the one-step glutaraldehyde procedure of Avrameas, Immunochem. 6:43 
(1969). 
EXAMPLE 6 
Therapeutic Abortion Test Production of Therapeutic Abortion Test 
Samples obtained during a therapeutic abortion are tested to verify that 
fetal materials have been removed from the uterus. 
Positive and negative controls are included in the test. The positive 
control, amniotic fluid having a known concentration of fetal fibronectin 
is diluted 1/10, 1/30, 1/90, 1/270, 1/710 and 1/2130 in Storage Solution. 
The Storage Solution consisting of 0.05M Tris-HCl, pH 7.4; 0.15M NaCl, 
0.02% NaN.sub.3, 1% BSA, 500 Kallikrein Units/mL of aprotinin, 1 mM 
phenylmethylsulfonyl fluoride (PMSF) and 5 mM EDTA. This Storage Solution 
is described in U.S. patent application U.S. patent application Ser. No. 
244,969, filed Sep. 15, 1988, the entire contents of which are hereby 
incorporated by reference. The negative control is first trimester 
maternal plasma diluted 1/5 and 1/10 in Storage Solution. Storage Solution 
is used as the negative background control. 
A sample of tissue or other material removed during a therapeutic abortion 
is dispersed in 0.75 mL of Storage Solution. The sample solutions are 
centrifuged in 1 mL microfuge tubes (13,000 rpm for 5 minutes) to remove 
particulate matter. 
A microtiter plate prepared as in Example 4 is used for the assay. 100 
.mu.L of each standard, sample, positive and negative control are placed 
in separate wells and incubated 2 hours at room temperature. The plate is 
washed 4 times with Wash Buffer, described in Example 3. 100 .mu.L of 
alkaline phosphatase-conjugated goat anti-(hCG) prepared as in Example 5 
is diluted 1/1000 in Conjugate Buffer (0.02M Tris-HCl, pH 8, 0.3M NaCl, 
0.05% TWEEN 20, 5% BSA, 0.02% NaN.sub.3). 100 .mu.L is dispensed into each 
well and incubated for 2 hours at room temperature. The plate is washed 4 
times as previously described. 4 mg/mL of p-nitrophenylphosphate (PNPP) is 
used as the substrate. This is diluted in 0.18M 
2-amino-2-methyl-1-propanol (AMP) buffer, pH 9.5 with 0.12 mM MgCl.sub.2. 
100 .mu.L is dispensed into each well of the microtiter plate. After a 5 
minute incubation at room temperature, the reaction rate in milli-OD/min 
is read at 405 nm on a V-MAX.TM. kinetic microtiter plate reader 
(Molecular Devices Corp., Palo Alto, Calif.). 
A standard curve is constructed by correlating increasing reaction rate 
with increasing fetal restricted antigen concentration in the standards. 
Unknowns are calculated directly from the curve or by using a pre-set 
computer program (Molecular Devices). 
In samples containing products of conception, significant amounts of fetal 
fibronectin are found, confirming the existence of normal pregnancy and 
the termination thereof. If, in a pregnant patient, significant amounts of 
fetal fibronectin are absent, the possibility of an unterminated ectopic 
pregnancy is indicated. 
EXAMPLE 7 
Spontaneous Abortion Test 
The procedure of Example 6 is repeated with samples of vaginal discharge 
obtained during a suspected spontaneous abortion of a normal pregnancy. 
Samples from a spontaneous abortion (miscarriage) demonstrate significant 
levels of fetal fibronectin in the discharge. If fetal fibronectin is not 
detected in significant levels in the discharge, a continuing pregnancy is 
indicated.