U.S. patent application number 13/366200 was filed with the patent office on 2012-05-31 for diagnostic method for determining the susceptibility to delivery and reagent kit for use thereof.
This patent application is currently assigned to Oy Medix Biochemica AB. Invention is credited to Eeva-Marja RUTANEN.
Application Number | 20120135432 13/366200 |
Document ID | / |
Family ID | 8535548 |
Filed Date | 2012-05-31 |
United States Patent
Application |
20120135432 |
Kind Code |
A1 |
RUTANEN; Eeva-Marja |
May 31, 2012 |
DIAGNOSTIC METHOD FOR DETERMINING THE SUSCEPTIBILITY TO DELIVERY
AND REAGENT KIT FOR USE THEREOF
Abstract
The invention relates to a diagnostic method for detecting
susceptibility to delivery, and to a test kit for this purpose. A
low, but higher than baseline level concentration of Insulin-like
Growth Factor Binding Protein 1 (IGFBP-1), which is due to leakage
from decidual cells, is detected by an immunological assay in a
vaginal secretion sample.
Inventors: |
RUTANEN; Eeva-Marja; (Espoo,
FI) |
Assignee: |
Oy Medix Biochemica AB
Kauniainen
FI
|
Family ID: |
8535548 |
Appl. No.: |
13/366200 |
Filed: |
February 3, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12498830 |
Jul 7, 2009 |
|
|
|
13366200 |
|
|
|
|
11048685 |
Jan 31, 2005 |
|
|
|
12498830 |
|
|
|
|
08360815 |
Dec 29, 1994 |
|
|
|
PCT/FI93/00275 |
Jun 29, 1993 |
|
|
|
11048685 |
|
|
|
|
Current U.S.
Class: |
435/7.92 ;
436/501 |
Current CPC
Class: |
G01N 33/689 20130101;
G01N 2800/368 20130101; Y10S 435/975 20130101; Y10S 436/814
20130101 |
Class at
Publication: |
435/7.92 ;
436/501 |
International
Class: |
G01N 21/78 20060101
G01N021/78; G01N 33/577 20060101 G01N033/577; G01N 33/566 20060101
G01N033/566 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 1992 |
FI |
923025 |
Claims
1. A method for determining the susceptibility to delivery and the
maturity of the cervix when foetal membranes of a pregnant woman
are intact, wherein the method comprises detecting a level of
insulin-like growth factor binding protein 1 (IGFBP-1) in a vaginal
or cervical secretion sample with an antibody that recognizes
IGFBP-1.
2. The method according to claim 1, wherein said antibody is a
monoclonal antibody.
3. The method according to claim 1, wherein said level of IGFBP-1
is determined by a quantitative, semiquantitative or qualitative
immunological assay.
4. The method according to claim 1, wherein said secretion sample
is obtained from the cervix.
5. The method according to claim 1, wherein the method comprises
detecting an IGFBP-1 level that is equal to or greater than a
normal baseline level of pregnancy.
6. The method according to claim 1, wherein said determined level
of IGFBP-1 is above about 1 .mu.g/l.
7. The method according to claim 1, wherein the method comprises
providing medical advice designed to prevent preterm delivery.
8. The method according to claim 1 for identifying a pregnant woman
possessing risk factors indicative of preterm delivery, wherein the
method comprises detecting a level of IGFBP-1 being equal to or
greater than said normal baseline level of pregnancy indicating
that said pregnant woman possesses risk factors indicative of
preterm delivery, said risk factors of said pregnant woman being
increased as compared to pregnant women with an IGFBP-1 level below
said normal baseline level of pregnancy.
9. The method according to claim 1 for assessing a time for
induction of labor, wherein the method comprises initiating
induction of labor for a pregnant woman where said sample, despite
intact foetal membranes, has an IGFBP-1 level that is equal to or
greater than a normal baseline level of pregnancy, wherein said
level of IGFBP-1 being equal to or greater than said normal
baseline level of pregnancy indicates maturation of the cervix of
said pregnant woman and impending delivery.
10. The method according to claim 9, wherein said time of induction
is assessed in pregnant women with a post-term pregnancy.
11. The method according to claim 9, wherein said time of induction
is assessed in pregnant women with a risk of pre-term delivery.
12. The method according to claim 1, wherein the method comprises
detecting that the foetal membranes are intact by examination.
13. The method according to claim 1, wherein the method comprises
detecting that the foetal membranes are intact by a diagnostic
method detecting a level of IGFBP-1 in a vaginal or cervical
secretion sample from a pregnant woman with an antibody that
recognizes IGFBP-1 and wherein said sample has an IGFBP-1 level
that is below a high IGFBP-1 level caused by the presence of
amniotic fluid resulting from rupture of a foetal membrane.
14. The method according to claim 13, wherein said high IGFBP-1
level is above about 100 .mu.g/l.
Description
[0001] This application is a Continuation of U.S. application Ser.
No. 12/498,830 filed on Jul. 7, 2009, which is a Continuation of
U.S. application Ser. No. 11/048,685 filed on Jan. 31, 2005, which
is a Continuation of co-pending application Ser. No. 08/360,815
filed on Dec. 29, 1994 and for which priority is claimed under 35
U.S.C. .sctn.120. application Ser. No. 08/360,815 is the national
phase of PCT International Application No. PCT/FI93/00275 filed on
Jun. 29, 1993 under 35 U.S.C. .sctn.371. The entire contents of
each of the above-identified applications are hereby incorporated
by reference. This application also claims priority of Application
No. 923025 filed in FINLAND on Jun. 29, 1992 under 35 U.S.C.
.sctn.119.
[0002] The present invention relates to a diagnostic method for
determining susceptibility to delivery based on a determination of
a protein in a secretion sample taken from the vagina or the
uterine cervix of a pregnant woman, and to a test kit designed for
this purpose. Determination of susceptibility to delivery is
particularly important in terms of the appropriate timing of
induction of delivery when the pregnancy is post-term, or when a
risk of preterm delivery is suspected.
[0003] Post-term pregnancies are a common yet difficult problem in
connection with delivery. A pregnancy exceeding 41-42 weeks is
considered post-term. Post-term pregnancies account for about 7-12%
of all pregnancies, the duration of the pregnancy being calculated
from the first day of the last menstrual period. About 4% of all
pregnancies continue over 43 weeks. Diagnosis is difficult, even
when the first day of the last menstrual period is known. Post-term
pregnancy is associated with increased foetal and neonatal
morbidity and mortality.
[0004] Perinatal mortality increases after the 42nd week of
gestation, and doubles after the 43rd week.
[0005] Monitoring the well-being of the foetus is vital in cases of
post-term pregnancy; delivery is, of course, immediately induced if
there are any signs of foetal distress. If, on the other hand, the
foetus is well, the next step is to decide the appropriate time for
induction. It is generally accepted that labour should be induced
as soon as the cervix is "mature" (shorter, softer and possibly
partly dilated). Attempts to induce labour too early, i.e. when the
cervix is not mature, lead to an increase in the number of
sections. On the other hand, if the status of the foetus requires
rapid delivery and it is known for sure that the cervix is not
mature, the decision will be to operate right away.
[0006] The maturity of the cervix is usually determined using the
so-called Bishop's score (Bishop E H, Obstet. Gynecol. 1964; 24:
266). The Bishop method involves assessing, by palpation, the size
of the cervical orifice and the length, softness and direction of
the cervical canal. The prior art method for determining the
maturity of the cervix is not always sufficient for reliably
predicting the time of delivery or for the timing of the induction
of labour and estimating the likelihood of success.
[0007] Attempts to induce dilation of the cervix are not always
successful. Prolonged labour increases the risk of maternal and
foetal infection and the risk of foetal asphyxia, as well as being
a psychologically unpleasant experience for the mother. Methods for
more accurate assessment of the maturity of the cervix are
therefore needed.
[0008] The present invention uses a completely new approach for
predicting the time of delivery. The maturity of the cervix is
assessed by determining the level of IGFBP-1, Insulin-like Growth
Factor Binding Protein 1, produced by decidual cells, said
assessment being performed on a vaginal or cervical secretion
sample taken from a pregnant woman.
[0009] As the cervix matures and painless uterine contractions
increase, the decidua, i.e. the endometrial lining of the mother's
uterus and the chorion attached thereto start to become detached
from each other in the lower segment of the uterus. When this
happens, decidual cells become damaged and compounds synthesized by
said cells are likely to "leak" from the cervix into the
vagina.
[0010] The inventor of the present invention realized that a low
but higher than a normal baseline IGFBP-1 concentration in a
vaginal or cervical secretion is indicative of such "leakage" and
is related to the maturation of the cervix and thus indicates an
impending delivery. If no such maturation takes place in the lower
segment of the uterus, the chorion remains attached to the decidua
and no decidual cell product is found in said secretion.
[0011] Determination of IGFBP-1 can therefore function as a
biochemical test for assessing the maturity of the cervix and thus
the time of delivery, or for deciding when induction should be
started. Said test can also be used to identify women in whom
induction probably would not help to dilate the cervix, thus
helping to prevent prolonged induction attempts, with the attendant
risks to both foetus and mother.
[0012] Another situation where a correct estimate of the time of
delivery is critical, is when a preterm delivery is suspected.
Predicting a preterm delivery is difficult. Identification of the
risk factors involved helps to predict only a very small proportion
of preterm deliveries.
[0013] Predicting a preterm delivery on the basis of uterine
contractions is often difficult. Preterm contractions may be so
weak that neither the woman nor the attendant nurse or physician
will notice them. On the other hand, out of the women who feel
preterm contractions and whose contractions are also objectively
verifiable, only about 10-20% are believed to need treatment to
prevent a possibly impending preterm delivery. It is difficult to
identify these 10-20%. However, in order to minimize the number of
preterm deliveries, it would be important to be able to identify
such patients as early as possible, i.e. as soon as contractions
start appearing.
[0014] Patients having any of the risk factors indicative of
preterm labour should be especially closely monitored. In ideal
cases, after a positive result in a test predicting preterm labour,
a preterm delivery could be better prevented by medication and
changes in lifestyle. Diagnosed preterm rupture of foetal membranes
and vaginal bleeding are as such signs of impending preterm labour,
and laboratory tests are not needed in such cases. However, if the
foetal membranes are intact, only regular contractions and observed
changes in the cervical canal (shortening of the canal and opening
of the orifice to 2 cm or more) are generally considered diagnostic
criteria for an impending preterm delivery and indications for
hospital admission. This may, in certain cases, prove to be too
late.
[0015] Several scoring systems have been developed for predicting
the risk of preterm labour. The scores are calculated during the
first pregnancy examination and used later for purposes of
comparison. However, these prior art systems only help to chart
about half of the pregnancies that end in preterm delivery. On the
other hand, based on said scoring systems, many women who do not
deliver their babies preterm end up in the risk group. Attempts
have been made to devise biochemical tests (e.g. determination of
blood oestradiol, blood progesterone and prostaglandin levels) but
with poor results.
[0016] The methods used so far have not provided a reliable basis
for predicting the significance of preterm contractions and thus
the likelihood of preterm labour. Therefore, it has not been
possible to direct prophylactic treatment at the right group of
pregnant women. In some women, preterm contractions are so weak
that they remain unnoticed until the labour is so advanced and the
cervix so dilated that the foetal membranes rupture. The only
choice is to treat those women who already have clinically
observable contractions. However, only in certain cases do preterm
contractions lead to preterm labour, and thus only this portion of
the women who have clinically confirmed preterm contractions need
prophylactic treatment (rest and tocolytic agents, i.e. agents
inhibiting uterine contractions). On the other hand, it has been
found that tocolytic treatment is ineffective and that preterm
labour cannot be prevented if considerable changes have already
taken place in the cervix before the medication is started.
[0017] The method according to the present invention, wherein the
amount of IGFBP-1 in a vaginal or cervical secretion sample is
determined, can be used in the prediction of preterm labour.
[0018] The protein IGFBP-1 is a product of the decidual cells. It
is found in the vaginal secretion of non-pregnant women only during
menstruation. Urine and seminal plasma, which may be present in the
vagina of pregnant women, contain only very low concentrations of
IGFBP-1.
[0019] The inventor of the present invention, Eeva-Marja Rutanen,
has previously shown that rupture of the foetal membranes can be
confirmed by determining the IGFBP-1 concentration in a vaginal
secretion. International Patent Application PCT/FI91/00413 is based
on said observation. It was also shown that vaginal secretion may
contain small amounts of IGFBP-1 even if the foetal membranes are
intact.
[0020] It can generally be assumed that biological membranes such
as foetal membranes may allow the passage of small amounts of
compounds and leak fluids such as amniotic fluid into the vagina
even if the membranes are intact. This would result in steady or
occasional low concentrations of IGFBP-1 in the vaginal or cervical
secretion. The inventor of the present invention realized that a
rising but low IGFBP-1 level does not represent the baseline level
that would result from amniotic fluid leaking through intact
membranes. The IGFBP-1 present in the secretion is not a result of
ruptured membranes either, since its level is low; instead it
originates from decidual cells in which some change is taking
place. This change is the detachment of foetal membranes from each
other which occurs before labour.
[0021] Low IGFBP-1 concentrations probably result from leakage from
decidual cells in the lower segment of the uterus, which may happen
when the decidual membrane and the chorion become detached from
each other as uterine contractions increase. A high IGFBP-1
concentration in the secretion therefore indicates the presence of
amniotic fluid, i.e. rupture of the membranes, whereas low
concentrations indicate protein secretion from the decidual cells
into the cervical canal and changes in the chorion-decidual border,
which in turn reflects subclinical changes in the cervix.
[0022] The use of a protein (foetal fibronectin) produced by
chorionic cells to indicate the rupture of foetal membranes is
described in European Patent Application EP 316919. Said
Application discloses that high fibronectin levels indicate a
rupture of the membranes, thus indicating impending preterm labour
(see also Lockwood C J, Senyei A E, Dische M R et al., New Engl. J.
Med. 1991; 325: 669). However, the disclosed method has the
disadvantage that the concentration of foetal fibronectin is high
in seminal plasma, which represents a major source of error.
Another factor that lowers the value of said method is that the
ratio between foetal fibronectin concentrations in serum and in
amniotic fluid is not as good as it is for IGFBP-1. This means that
there is a greater risk that an unnoticed small blood contamination
could interfere with the determination. A test was performed
wherein protein levels were determined from vaginal secretion in 20
women whose foetal membranes were intact and who did not have
uterine contractions. The fibronectin test gave four positive
results, whereas IGFBP-1 determination gave no positive
results.
[0023] The above European Patent Application does not suggest the
use of low fibronectin values for determining susceptibility to
delivery when foetal membranes are intact.
[0024] In situations where a test is used to predict the time of
delivery, it is valuable to obtain the test result rapidly. The
ideal test for this purpose would be simple and quick to perform,
preferably as a bed-side test, i.e. immediately on the site.
[0025] An object of the present invention is to provide a new and
improved method for determining the maturity of the cervix and the
risk of impending preterm labour, the method being specific to the
substance to be measured independent of individual patient
variations.
[0026] It is also an object of the present invention to provide a
method for determining the susceptibility to delivery, the method
being rapid and simple to perform, preferably while the patient is
waiting (bed-side test).
[0027] Another object of the invention is to develop a test kit
suitable for such a diagnosis, the kit containing the means for
performing a simple and rapid diagnostic test.
[0028] The details of the present invention will be evident from
the following description and the appended claims, the contents of
which are included herein by this reference. Thus, the present
invention concerns a diagnostic method for determining
susceptibility to delivery, said method being based on the
determination of a protein in a vaginal or cervical secretion
sample of a pregnant woman. According to the invention, leakage of
IGFBP-1 from decidual cells is determined by detecting, in an
essentially blood-free secretion sample, that the IGFBP-1
concentration has increased to a level which is higher than the
normal baseline level during pregnancy, but still lower than the
high IGFBP-1 level caused by the presence of amniotic fluid due to
a rupture of the foetal membranes. The presence of IGFBP-1 in the
sample is best shown using an IGFBP-1-specific binding substance or
substances, preferably an IGFBP-1 monoclonal antibody.
[0029] The test kit for the diagnosis of susceptibility to delivery
according to the present invention includes at least one reagent in
which there is an IGFBP-1 specific binding substance for showing an
elevated but still low IGFBP-1 level in a vaginal or cervical
secretion sample. The test kit according to the invention also
includes at least one label for indicating the binding reaction
between IGFBP-1 and said binding substance. The signal(s)
indicating the reagent binding reaction is/are chosen so that when
the IGFBP-1 concentration of said sample exceeds the normal
baseline level during pregnancy, a positive signal is obtained to
indicate the binding reaction, and when the IGFBP-1 concentration
exceeds the high IGFBP-1 level caused by amniotic fluid, another
positive signal is obtained.
[0030] The preferred specific binding substance for IGFBP-1 is a
specific antibody of IGFBP-1, particularly a monoclonal
antibody.
[0031] The protein IGFBP-1 was first isolated from the placenta and
foetal membranes in 1980 (Bohn et al., Arch. Gynecol. 1980; 229:
279-291). It was considered to be a protein of placental origin, it
was given the name Placental Protein 12 (PP12). Later, it was
observed that PP12 and IGFBP-1 isolated from amniotic fluid had the
same N-terminal amino acid sequence and that PP12 binds IGF I
(insulin-like growth factor I) (Koistinen et al., Endocrinology
1986; 118: 1375). Synthesis of IGFBP-1 in the decidua of a pregnant
woman was first demonstrated in 1985 (Rutanen et al., Endocrinology
1985; 116: 1304).
[0032] The concentration of IGFBP-1 in amniotic fluid has been
found to be usually 100 to 1000 times higher than that in maternal
serum (Rutanen et al., Am. J. Obstet. Gynecol. 1982; 144:460). The
clinical application of this finding is the basis for Patent
Application PCT/FI91/00413 (priority date Dec. 30, 1990) assigned
to the same applicant. According to said invention a suitably
unsensitized test is used for detecting the presence of IGFBP-1 in
the vagina caused by a rupture of the foetal membranes. Detecting
the rupture of the foetal membranes using IGFBP-1 is also described
in an article (Rutanen E-M, Pekonen F., Am. J. Obstet. Gynecol.
1991; 164 (Suppl): 258).
[0033] The presence of IGFBP-1 after rupture of the foetal
membranes is basically a single event involving a sharp, clear
elevation, whereas decidual leakage of IGFBP-1 and its presence in
the secretion are characterized by gradual elevation as the time of
delivery approaches, i.e. as the cervix matures, an event that
never leads to very high concentrations.
[0034] The reactions based on specific binding substances are
widely known. Antibodies are the compounds most commonly used as
specific binding substances. These so-called immunological assays
are based on the ability of an antibody to bind specifically to a
certain site in its antigen (epitope). Polyclonal antibodies are a
mixture of immunoglobulins present in the serum of an immunized
animal. The mixtures vary between individual animals. Monoclonal
antibodies, on the other hand, are produced by one cell line
cultured in a laboratory. Such antibodies are homogeneous and can
be characterized by methods used in protein chemistry; they can be
produced continuously in identical form.
[0035] An immunological assay can involve the use of only one
antibody. In this case, the reaction conditions are usually chosen
so as to allow the antigen in the test sample to compete with an
added labelled antigen for a limited number of binding sites in the
antibody. Except for said label, said added antigen is identical
with the test sample antigen. The concentration of the sample
antigen is obtained by determining the fraction of bound label.
Several labelling substances that produce a signal enabling
measurement of concentration may be used, e.g., radioactive
isotopes, enzymes, and chemiluminescent or fluorescent compounds.
The method may also employ two different antibodies (the so-called
sandwich principle). Here, the antibodies are specific to separate
epitopes in the same antigen, and can bind simultaneously to the
same antigen molecule. One antibody is usually immobilized on a
solid carrier and the other one is labelled. Both antibodies bind
to the antigen in the sample, and the complex can be separated from
unbound label with the aid of said carrier. The amount of bound
labelled antibody is directly proportional to the antigen
concentration in the sample.
[0036] The present inventor has studied placental proteins, and for
her own research work she has developed a radioimmunological assay
method for detecting IGFBP-1 (PP12). Monoclonal anti-bodies to
IGFBP-1 have also been developed for research purposes (Rutanen et
al., Biochem. Biophys. Res. Commun. 1988; 152: 208 and Pekonen et
al., J. Immunoassay. 1989; 10: 325-337).
[0037] No report has been found in the literature showing a
comparison between the concentrations of IGFBP-1 in amniotic fluid
and in other secretions present in the vagina. A research group led
by the present inventor has studied the concentrations of IGFBP-1
in amniotic fluid and, blood (Table 1), as well as the
concentrations in secretions that can be present in the vagina.
TABLE-US-00001 TABLE 1 Concentrations of IGFBP-1 in maternal serum
(S) and amniotic fluid (AF) in samples taken at gestation of 24-38
weeks. IGFBP-1 IGFBP-1 IGFBP-1 ratio Patient S (.mu.g/l) AF
(.mu.g/l) AF/S 1 170 60000 353 2 130 29000 223 3 140 48000 343 4
200 65000 325 5 63 22000 349 8 350 350000 1000 7 240 115000 479 8
190 40000 210 9 240 50000 208 10 250 200000 800 11 130 70000 538 12
65 33000 508 13 600 70000 117 14 260 95000 365 15 340 180000 529 16
500 155000 310 17 155 19000 122 18 340 140000 412 19 160 200000
1250 20 240 160000 667 21 340 145000 426 22 350 53000 151 23 200
125000 625 24 135 55000 407 25 340 145000 426
[0038] In the paired blood and amniotic, fluid samples studied, the
concentration of IGFBP-1 in the amniotic fluid was in all cases
more than 100 times higher than in maternal serum.
[0039] It has also been shown that vaginal secretions as such,
sperm and urine contain almost no IGFBP-1. The test according to
the present invention can thus be designed so that the presence in
the sample of such secretions does not cause false positive results
in test situations.
[0040] According to the inventor's observations, the concentration
of IGFBP-1 in vaginal or cervical secretions can be elevated even
when the foetal membranes are intact. However, in a sample
extracted in 0.5 ml of a buffer, the IGFBP-1 concentration does not
exceed about 100 .mu.g/l, unless it originates from amniotic fluid
resulting from ruptured foetal membranes. This finding has brought
to light the fact that, when the foetal membranes remain intact,
this slight but higher than baseline elevation can be interpreted
as protein leaking directly from decidual cells. If the sample
contains amniotic fluid, the test based on the invention cannot be
used to show elevation of the IGFBP-1 level due to leaking decidual
cells, since the concentration of IGFBP-1 due to the presence of
amniotic fluid is higher. Rupture of the foetal membranes can be
detected by examination, by quantitative determination of IGFBP-1
or by diagnostic methods such as the one mentioned above (Patent
Application PCT/FI91/00413).
[0041] Table 1 shows that in pregnant women, the serum
concentration of IGFBP-1 can be as high as 600 .mu.g/l. It is
therefore evident that a secretion sample contaminated by blood is
not well suited for the test according to the invention for
predicting the time of delivery. However, this does not represent a
diagnostic problem because even a slight bleeding from the cervical
canal is considered a risk factor associated with preterm labour.
If the bleeding should stop, cervical maturation can be monitored
by determining the concentration of IGFBP-1 as described in the
present invention.
[0042] Examples of situations where the concentration of IGFBP-1 in
the vaginal secretion has been monitored during pregnancy are shown
in Table 2. Table 3 shows individual cases representing
concentrations of IGFBP-1 in extracted cervical secretion in
various situations. The IGFBP-1 concentrations shown in said tables
were obtained using secretion samples extracted in 0.5 ml of buffer
and the quantitative test IGFBP-1 IEMA Test Cat 10831 (Oy Medix
Biochemica Ab, Finland).
TABLE-US-00002 TABLE 2 Gestation IGFBP-1 Contrac- Patient weeks +
days .mu.g/l tions Remarks 1 30 + 1 0 + delivery week 41 + 3 32 + 2
0 (+) 35 + 1 0 + 37 + 1 0 2 37 + 1 0.6 - delivery week 41 + 3 39 +
4 0 - post-term pregnancy 41 + 2 0 - membranes punctured 3 31 + 5 0
+ 33 + 4 0 - 36 + 4 0 + 38 + 3 0 (+) 40 + 2 0 4 24 + 1 0.6 + 25 + 1
1.8 (+) 27 + 2 0 (+) 29 + 2 0 (+) 32 + 2 0 - 5 28 + 5 0 + delivery
week 38 + 4 30 + 4 0 (+) emergency section 32 + 4 0 - foetus
transverse 34 + 4 2.3 (+) twin pregnancy 36 + 3 0 - 6 32 + 3 0 -
delivery week 40 + 2 36 0 (+) 39 + 3 3.9 (+) 7 25 + 3 0 + delivery
week 35 + 2 27 + 5 0 (+) emergency section 29 + 4 0 (+) twin
pregnancy 31 + 5 0 (+) 33 + 6 8.8 (+) 8 32 + 2 0 - delivery week 42
+ 2 36 + 2 0 - 40 + 2 0 - 41 + 6 2.5 - 9 24 3.0 + delivery week 33
26 0 (+) section 28 0 (+) triple pregnancy 30 0 - 31 + 1 1.8 + 10
29 + 6 0.6 (+) delivery week 33 + 6 30 + 5 0 - section 31 + 5 5.3 -
triple pregnancy
TABLE-US-00003 TABLE 3 Pa- Gestation IGFBP-1 Contrac- Mem- tient
weeks + days .mu.g/l tions branes Delivery A 41 + 3 0 - intact
induction B 35 8100 - ruptured 35 + 1 spont. C 41 + 2 0.7 - intact
41 + 6 spont. D 41 + 5 6160 - ruptured 41 + 6 spont. E 39 61 intact
39 + 1 spont. F 39 + 2 0 - intact induction G 35 1 (+) intact 35 +
6 twins H 35 13 - intact 35 + 6 I 31 + 5 0.4 - intact 40 (spont. =
spontaneous)
[0043] From the data shown in Tables 2 and 3, it can be concluded
that the IGFBP-1 level in an extracted secretion sample is normally
below the limits of detection of the method, i.e. below about 0.5
.mu.g/l. This can therefore be considered the baseline level.
Concentrations above the baseline level indicate the presence of
some active process. This, together with clinical information, is
used to decide whether any treatment is necessary.
[0044] Table 3 shows that, as spontaneous, delivery approached in
women whose membranes were still intact, the concentration of
IGFBP-1 in the extracted sample varied between about 1 and 61
.mu.g/l. Higher concentrations (8100, 6160 .mu.g/l) associated with
ruptured foetal membranes are clearly distinguishable.
[0045] In practice it is difficult to set a definite lowest limit
for a clinically significant IGFBP-1 concentration, because many
factors affect sampling. This is why the lowest limit of the test
should be set to a value just above the baseline level, e.g. 1
.mu.g/l, as the aim is to detect rising values.
[0046] A small amount of secretion (100-200 .mu.l) is sufficient
for the test. The sample can be taken during a speculum
examination, e.g., into a syringe or with a sterile sampling device
made for this purpose. In practice, the simplest method is to
absorb the secretion into a swab and then extract it into a buffer
solution.
[0047] Since it is difficult to obtain a specific amount of vaginal
or cervical secretion, the samples mentioned in this patent
application were extracted into 0.5 ml of buffer, and the IGFBP-1
concentrations mentioned in the description and in the appended
claims refer to results obtained from this solution. The IGFBP-1
concentration of the native sample is therefore about five times
the concentrations given.
[0048] Despite the inaccuracy of sampling, a reliable diagnosis can
be obtained since the elevation of the IGFBP-1 concentration is so
clear that variations in sampling do not greatly affect
interpretation of the results.
[0049] The concentration of IGFBP-1 is best determined using an
immunological assay, which may be quantitative, semiquantitative or
qualitative. In the preferred qualitative embodiment, the
measurement range is such that even a slightly elevated
concentration of IGFBP-1 is clearly differentiated as positive from
the low normal baseline level that is the negative result. In such
a case, a first positive signal is obtained when the concentration
of IGFBP-1 is over about 0.5 .mu.g/l, preferably over 1 .mu.g/l,
and a second positive signal is obtained when the concentration of
IGFBP-1 is about 100 .mu.g/l or over.
[0050] A semiquantitative determination can be made, for instance,
using several reagents indicating different IGFBP-1 concentrations,
a positive signal occurring when the IGFBP-1 concentration exceeds
predetermined cut-off values such as 1 .mu.g/l, 2.5 .mu.g/l, 5
.mu.g/l, 10 .mu.g/l etc.
[0051] The preferred IGFBP-1 test according to the present
invention has been developed to give a result as rapidly as
possible, which is advantageous both medically and economically in
establishing the diagnosis intended in the invention. The sample
required can be taken during a gynaecological speculum examination.
The sample can also be taken using a swab, into a syringe or with a
sampling device made for this purpose.
[0052] When binding substances specific to IGFBP-1 only are used,
false positive reactions caused by a cross-reaction, i.e. binding
of the wrong compound, are eliminated.
[0053] According to the present invention, the IGFBP-1 test is
preferably performed using two specific monoclonal antibodies, e.g,
so that one is attached to a small plastic bead and the other one
is coupled to a label, for example an enzyme, such as horseradish
peroxidase (HRP). The sample, the enzyme-labelled antibody and the
antibody-coated bead in a gripper are placed in a test tube. When
the mixture is incubated, the IGFBP-1 present in the sample will
become attached both to the bead and to the labelled antibody.
After incubation, the bead is removed from the tube and washed
under running water. The bead is placed in a tube containing the
substrate solution of the enzyme used as label. During incubation,
a visible color develops if the sample contains a sufficient amount
of IGFBP-1. The solution remains colorless if the sample does not
contain IGFBP-1, or if its concentration is too low. A quantitative
value is obtained for the IGFBP-1 concentration if the absorbance
of the solution is determined photometrically.
[0054] The IGFBP-1 test according to the present invention can also
be performed by attaching the first antibody to the surface of a
membrane developed for such test purposes. The sample is placed in
contact with the membrane, and the IGFBP-1 in the sample will bind
specifically to the immobilized antibody. A corresponding
enzyme-coupled antibody is then added, which in turn binds to the
IGFBP-1 now present on the membrane. The membrane is washed and
bound enzyme is detected by adding to the washed membrane a
precipitating substrate of the enzyme, which changes color in the
presence of said enzyme. Thus, when the sample is positive, a
visible color develops on the membrane. This kind of test, which is
based on an antibody-coated membrane, can be provided, for
instance, by attaching such a membrane to a plastic vessel
especially designed for the purpose. An absorbent material placed
under the membrane will rapidly absorb the test solutions through
the membrane when the liquids are pipetted onto the membrane. The
membrane can also be attached to a plastic strip, which is then
transferred from one solution to another.
[0055] A color indicating a positive result can also be obtained
otherwise than by labelling the antibody with an enzyme, which in
turn causes said change in the color of its substrate. Instead of
labelling the antibody with an enzyme, a dye can be used to produce
a color indicating a positive result. The intensity of the color of
the label bound to the immobilized IGFBP-1 in a positive test
situation should be strong enough to be visible. Gold or selenium
colloids or disperse dyes can be used for this purpose. The
advantage of such dyes is that the test is quicker to perform since
a separate substrate reaction phase is not needed. Correspondingly,
the antibody can be coupled to colored latex particles. When latex
or dye particles are used for direct visual detection a rapid
immune-chromatographic test method can be used for IGFBP-1.
Typically, a membrane is used, and a first antibody is attached to
a small area of the membrane. A second color-labelled antibody is
attached to another area by drying. The second antibody starts to
migrate across the membrane when a liquid sample is added. If the
sample contains a sufficient amount of IGFBP-1, a colored zone will
develop at the point where the antigen bound to the labelled
antibody binds also to the antibody immobilized onto the membrane.
If the sample is negative, no colored zone develops, and the dye
migrates over the membrane.
[0056] An IGFBP-1 test can also be performed using the
agglutination principle. Here, the visible reaction takes the form
of an agglutination of, say, antibody-coated particles such as
latex, the antigen in the sample causing bonds to be formed between
the particles. Alternatively, detection can be based on the
inhibition of agglutination.
[0057] In addition to specific antibodies, methods and test kits
according to the present invention can make use of other specific
IGFBP-1 binding substances as well as combinations of the two. This
allows the natural binding characteristics of IGFBP-1 to substances
like IGF (Insulin-like Growth Factor) to be exploited.
[0058] The test can also be performed using the assay methods set
out in FI patent 84863, the contents of which are included herein
by reference.
[0059] The IGFBP-1 test methods according to the present invention
can greatly ease the problems related to assessing the time of
delivery. In post-term pregnancies the invention makes it possible
to reduce maternal and foetal morbidity by determining the optimal
time for induction of labour. This, in turn, will reduce the number
of cesarean sections. It will also make it easier to decide on an
emergency section as the primary mode of delivery in cases in which
the cervix does not show any signs of dilation. Excluding neonatal
malformations, preterm labour is the main reason for neonatal
morbidity and mortality. The problem can be considerably alleviated
by prophylactic treatment if this can be given early enough to the
right patients.
[0060] The decisions outlined above are significant both
economically and medically: economically in terms of the cost of
hospitalization, and medically in terms of greater maternal and
foetal well-being and lower mortality.
[0061] The test kit according to the present invention contains a
reagent based on a specific binding substance for IGFBP-1.
Depending on the test method employed, the reagent may be a binding
substance solution, a solid phase like a bead or membrane coated
with a specific binding substance, or latex or dye particles. For
example, in a one-step assay, the kit may also contain a
combination of the above mentioned components. The specific binding
substance should preferably be a monoclonal antibody specific to
IGFBP-1.
[0062] In addition to the reagent mentioned above, the kit
preferably contains a label that is able to detect a sufficient
concentration of IGFBP-1 in the sample after the binding reaction.
The label detecting the binding reaction should preferably be a
signal-producing label coupled to another antibody to IGFBP-1. The
label which is coupled to another antibody to IGFBP-1 may be an
enzyme, a radioactive isotope or a compound that can be recognized
by its color. If the label is an enzyme, the test kit preferably
also contains a suitable substrate.
[0063] In addition to the essential reagents, the test kit
preferably also contains a solution for handling the sample (e.g.
for extracting the sample from the swab). A suitable solution would
be an extraction buffer, e.g., a phosphate buffer containing a
protective protein and having a pH close to the physiological
pH.
[0064] The test kit may also contain a sampling instrument, such as
a sterile swab, a disposable syringe or a device specially
developed for the test. If the result is to be quantified, the kit
may also contain the relevant standards.
[0065] The test kit may also comprise a simple device for taking a
vaginal secretion sample and a suitable buffer solution for
performing the test. The test kit may also comprise an
antibody-coated test strip that the patient herself can insert into
the vagina.
[0066] The preferred test kit according to the present invention is
a combination kit, allowing two tests to be performed
simultaneously on one sample. One test will assess the maturity of
the cervix, while the other one will detect rupture of the foetal
membranes, e.g., as described in Patent Application PCT/FI91/00413.
Such a combination kit could be based on the immunochromatographic
principle in which case it will comprise a membrane to which are
attached two zones of an IGFBP-1 binding substance. Said zones are
of different strength. One zone is used to indicate a low
concentration of IGFBP-1 in the sample, which in turn indicates
maturity of the cervix. If the concentration is high enough to
indicate the presence of amniotic fluid, a color reaction will
develop in both zones. Different dilutions of the label can also be
used, or different colored labels may be used, positive test
results being indicated by zones of different colors.
[0067] The test may also comprise several zones that give a
positive signal when the concentration of IGFBP-1 exceeds cut-off
values such as 1 .mu.g/l, 5 .mu.g/l, 10 .mu.g/l and 100
.mu.g/l.
[0068] The Examples below illustrate how the test according to the
invention can be carried out without, however, limiting it in any
way.
EXAMPLE 1
[0069] A sample was taken from the vagina of a pregnant woman
(gestation week 39+3) using a Dacron swab. The swab was held in the
vagina for about 15 seconds to allow a sufficient amount of
secretion to be absorbed into said swab. The sample was extracted
into 0.5 ml of an assay buffer (buffer solution containing a
stabilizer and a surfactant). The extracted sample was centrifuged
and the concentration of IGFBP-1 was determined quantitatively from
the supernatant (IGFBP-1 IEMA TEST Cat 10831-ETMB, manufactured by
Oy Medix Biochemica Ab, Finland).
[0070] The concentration of IGFBP-1 in the supernatant was 3.9
.mu.g/l, indicating that the cervix was mature. The patient gave
birth six days later.
EXAMPLE 2
[0071] Plastic beads are coated with IGFBP-1 antibody (6305, Medix
Biochemica). Another IGFBP-1 antibody (6303, Medix Biochemica) is
coupled with an enzyme label (horseradish peroxidase, HRP).
Phosphate buffer (pH 7.4) containing 0.3% bovine serum albumin
(BSA) is used as assay buffer. The buffer also contains a detergent
and preservatives. A rapid IGFBP-1 test is performed according to
the following instructions.
Performing the Rapid IGFBP-1 Test:
[0072] 1. 200 .mu.l of 6303-HRP label (diluted 1:50 in assay
buffer) are pipetted into a sample tube. 3. 100 .mu.l of sample
(extracted into 0.5 ml of buffer) are added to the tube. 4. An
IGFBP-1 antibody-coated bead in a gripper is placed into the tube.
It is incubated for 5 minutes. 5. The bead and the gripper are
removed from the solution and the bead is washed under running
water for 30 seconds. 6. The washed bead including the gripper are
transferred to 400 41 of substrate solution
(2,2'-azino-di-(3-ethyl-benzthiazoline sulfonate(6)], ARTS) in a
clear tube. 7. The tube is allowed to stand in a dark protected
bottle for 5 minutes. 8. The color of the solution is either
inspected immediately or the reaction is stopped by adding an
inhibitor solution (200 .mu.l) and then inspecting the solution: a
colorless solution indicates a negative result and green a positive
result.
EXAMPLE 3
[0073] A narrow zone of a nitrocellulose membrane strip is coated
with an IGFBP-1 antibody (6305, Medix Biochemica). Coloured latex
particles are coated with another IGFBP-1 antibody (6303, Medix
Biochemica). The coated latex particles are dried onto one end of
the membrane strip containing the antibody zone. A rapid IGFBP-1
test is performed on the membrane according to the following
instructions.
Performing the IGFBP-1 Membrane Test
[0074] 1. A few drops of the sample are pipetted onto that part of
the strip holding the dried latex particles. 2. Incubation for a
few minutes allows the sample to migrate across the membrane. The
latex particles, together with the liquid, will move over the
antibody-coated zone to the other end of the strip. 3. The strip is
inspected. The presence of a colored zone indicates a positive
result.
EXAMPLE 4
[0075] A small area of a nylon membrane is coated with IGFBP-1
antibody (6305, Medix Biochemica). The coated membrane is placed on
a plastic cup-like vessel so that immediately beneath and in
contact with the membrane is an absorbent material (treated
cellulose). Another IGFBP-1 antibody (6303, Medix Biochemica) is
coupled to an enzyme label (horseradish peroxidase, HRP). A rapid
IGFBP-1 test is performed according to the following
instructions.
1. A few drops of the sample solution are pipetted onto the
membrane and the solution is allowed to absorb through the
membrane. 2. As much wash solution as the cup will hold (about 1
ml) is pipetted onto the membrane and the solution is allowed to
absorb through the membrane. 3. A few drops of the label solution
are pipetted onto the membrane and the solution is allowed to
absorb through the membrane. 4. About 1 ml of a wash solution is
pipetted onto the membrane and the solution is allowed to absorb
through the membrane. 5. A few drops of precipitating substrate of
the enzyme are pipetted onto the membrane and the solution is
allowed to absorb through the membrane. 6. The membrane is
inspected. A colored zone indicates a positive result.
[0076] The immunometric methods described above illustrate how the
test according to the present invention may be carried out. It will
be evident to the person skilled in the art that the methods may be
varied and modified within the above specification and the appended
claims without deviating from the scope of the invention.
* * * * *