JP3943575B2 - An assay method to determine membrane rupture in women at risk for imminent delivery - Google Patents

Description

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to a method for detecting an impending delivery and determining whether the membrane has ruptured in a woman at such risk. In particular, the present invention detects the biochemical marker for imminent delivery in a cervicovaginal secretion sample and identifies the cause of that risk by identifying the absence of IGFBP-1 in the sample Is directed to the measurement of imminent labor by determining membrane rupture.

DESCRIPTION OF THE PRIOR ART Measurement of impending preterm births is essential to increase neonatal survival of preterm infants. In particular, preterm neonates are responsible for more than half, and perhaps as much as 3/4, of neonatal morbidity and mortality without birth defects. Although tocolytic agents that can delay delivery were introduced 20-30 years ago, there is only a slight decrease in the incidence of preterm birth. The failure to observe a large decrease in the incidence of preterm birth is presumed to be due to misdiagnosis in premature labor and that the patient's symptoms have progressed so far that preterm delivery agents cannot successfully delay the delivery Is done. Traditional methods of premature birth diagnosis have high false-negative and false-positive error rates [Friedman et al, Am. J. Obstet. Gynecol. 104: 544 (1969)]. Furthermore, traditional methods of measuring preterm labor, especially in patients with clinically intact membranes, can require complex training or equipment [Garl et al, Obatet. Gynecol. 60: 297 (1982)], or can be invasive [Atlay et al, Am. J. Obatet. Gynecol. 108: 933 (1970)]. An early objective biochemical marker indicating an increased risk of preterm birth was needed.

  Fetal fibronectin is synthesized by the extravillus trophoblast when trophoblasts invade the mother's decidualized uterus. Insoluble fetal fibronectin is in the extracellular matrix of the placental bed. Soluble fetal fibronectin is found in amniotic fluid. Fetal fibronectin secretion into the subpartum cervical secretion may be due to membrane rupture when amniotic fluid is released into the subpartum or to release fetal fibronectin from the extracellular matrix within the placental bed (solubilization). ) Or by release of fetal fibronectin from trophoblast cells. The presence of fetal fibronectin in cervical secretions is a predictor of preterm birth.

  Insulin-like growth factor binding protein 1, IGFBP-1, another name for IGFBP-1 is pregnancy-related endometrial α-globulin (α-PEG) , And placental protein-12 (pp-12) [Waites et al, J. Clinical Endocrinology and Metab. 67: 1100 1986]) in late secretion in response to fetal implantation into the uterine wall What is synthesized by the endometrial stroma in the pre-decidual phase and by the pregnancy-induced maternal decidua is a more abundant amniotic fluid protein than fetal fibronectin. IGFBP-1 appears to be efficiently transported into the amniotic sac from the decidua. The level of IGFBP-1 in amniotic fluid increases with the length of gestation. By the third trimester (27 weeks), IGFBP-1 levels increase to about 20 mg / ml, and IGFBP-1 is one of the most abundant proteins in amniotic fluid.

  Lockwood et al [New Engl. J. Med., 325: 669-674 (1991)] reported that fetal fibronectin in cervical and vaginal secretions indicates pregnancy at risk for imminent delivery. The author hypothesized that damage to the fetal membrane could release fetal fibronectin into its neck and vagina, giving rise to biochemical markers. Fetal fibronectin is present in the secretions whose imminent delivery is either due to rupture of the membrane or initiation of delivery.

  Rutanen et al (American Journal of Obsterics and Gynecology 164 (Supplement, Part 2): 258, 1991) found that IGFBP-1 was present in women with ruptured membranes and therefore at risk for imminent delivery. reported. Like fetal fibronectin, IGFBP-1 is one of the most abundant proteins in amniotic fluid and is also present in the decidua, which constitutes another marker for preterm birth. It suggests that you can.

  If IGFBP-1 or any other effective marker is released at various points in the course of preterm birth, the marker could be used to assess the course of the disease.

SUMMARY OF THE INVENTION The present invention provides an assay for distinguishing patients with impending imminent delivery with an intact membrane from patients with membrane rupture. The method uses cervical vaginal secretion samples from pregnant patients measured as at risk for imminent delivery by detection of biochemical markers for imminent delivery in cervical vaginal secretion samples from patients. Obtaining and measuring the level of IGFBP-1 in the sample. If the level of IGFBP-1 increases, the patient has a membrane rupture. In the absence of IGFBP-1, the patient has an intact membrane.

  In a preferred embodiment, the method comprises obtaining a cervical vaginal secretion sample from a pregnant patient about 20 weeks after pregnancy and measuring the levels of fetal fibronectin and IGFBP-1 in the sample. Become. The presence of elevated fibronectin in the sample indicates an increased risk of imminent delivery. If the level of IGFBP-1 is elevated, the patient has a membrane rupture. In the absence of IGFBP-1, the patient has an intact membrane.

  In the most preferred embodiment, the fetal fibronectin / IGFBP-1 test is preferably performed on women at about 20 weeks of pregnancy and repeated at each prenatal visit (every 2-4 weeks). For patients whose fetal fibronectin assay results indicate an increased risk of preterm birth, testing the patient's IGFBP-1 level determines whether the membrane is intact. If the test for IGFBP-1 is negative, the patient can be treated to delay their pregnancy. The test for IGFBP-1 levels can be repeated on a daily basis during the course of treatment to confirm that the membrane remains intact. In patients with increased levels of IGFBP-1, the test shows that delivery cannot be delayed.

Detailed Description of the Invention The present invention is an assay that determines membrane rupture in patients with preterm birth and identifies patients who can receive treatment to delay labor. The method obtains a cervical vaginal secretion sample from a patient determined to be at risk for imminent delivery by detection of a biochemical marker for imminent delivery in a cervical vaginal secretion sample from the patient, And testing a cervical vaginal secretion sample from the patient for IGFBP-1. If the level of IGFBP-1 increases, the patient has a membrane rupture. If IGFBP-1 is not detected, the patient has an intact membrane. In a preferred embodiment, cervical vaginal secretion samples from pregnant patients are tested for both IGFBP-1 and its delivery marker. In the most preferred embodiment, the delivery marker is fetal fibronectin.

  The present invention also provides a kit comprising an anti-insulin-like growth factor binding protein 1 antibody and an imminent delivery marker, preferably an antibody specific for fetal fibronectin.

Patients to be tested This method can be used for any pregnant woman who can be at risk for imminent delivery. In one embodiment of the method, an assay for IGFBP-1 is performed from a patient who has been positively tested for the presence of a biochemical indicator of risk for imminent delivery to determine whether the membrane is intact. For cervical vaginal secretions. In a preferred embodiment where both markers are assayed in the same sample, the method can be performed on any pregnant patient that satisfies the criteria for the imminent delivery marker. For example, the presence of fetal fibronectin in a cervical vaginal secretion sample indicates imminent delivery after 20 weeks of gestation until delivery, regardless of whether delivery is early or after the period has elapsed. Only patients within the appropriate gestational age range for fetal fibronectin should be tested for the marker combination.

  In addition to screening any woman to determine if labor is imminent, patients to be tested for labor indicators should have clinically intact membranes within the high risk category for preterm birth. And preferably all women whose pregnancy is not progressing sufficiently to ensure the delivery of a healthy fetus. 90% of fetal morbidity and 100% of fetal mortality associated with preterm birth are for delivered fetuses before the 32-34 week gestation period. Therefore, a gestation period of 32 to 34 weeks is an important cut-off for fetal health, and women whose pregnancy is at least about 20 weeks and before 34 weeks in gestation period are tested It should be.

  In addition, there are a number of factors known to be associated with the risk of preterm birth. These factors include: multiple fetusgestations; dysfunctional cervix; uterine abnormalities; polyhydramnios; nulliparity; prior premature membrane rupture or preterm birth; preeclampsia Preeclampsia; first trimester vaginal bleeding; little or no prenatal care; and signs such as abdominal pain, low spinal pain, passage and contraction of uterine mucus. Any pregnant woman with a clinically intact membrane and one or more risk factors for preterm birth in a gestation period of 12 weeks or more should be tested for the entire risk period; ie, up to about 37 weeks. is there. Risk factors for spontaneous miscarriage include weight fetal abnormalities, abnormal placenta formation, uterine abnormalities and maternal infectious diseases, endocrine disorders, cardiovascular renal hypertension, autoimmunity and other immunological diseases, and nutritional disorders.

Sample This sample is obtained near the posterior fornix, ectocervix or external cervical os. The sample generally comprises a liquid and a particulate solid and can contain vaginal or cervical mucus and other vaginal or cervical secretions. Such samples are referred to herein and in the claims as cervicovaginal secretion samples. The sample is preferably removed by a cotton swab with a Dacron or other fiber tip. Alternatively, the sample can be obtained by a suction or lavage device. Calculations to calculate additional dilutions for any of the samples taken using the liquid can be made as part of the interpretation of the assay procedure.

After collection, the sample is transferred to a container suitable for storage and transport to the test laboratory. It is important to disperse the sample in the liquid in which the proteinaceous analyte is stored. This storage and transfer medium should minimize and preferably prevent a decrease in analyte level during storage and transport. Suitable solutions for storage and transfer are 0.05 M Tris-HCl, pH 7.4; 0.15 M NaCl, 0.02% NaN ₃ , 1% bovine serum albumin (BSA), 500 Kallikrein Units / ml aprotinin, 1% phenylmethylsulfonyl · U.S. Pat. No. 4,919,889, which consists of fluoride (PMSF) and 5 mM EDTA and was granted on April 24, 1990. This solution is also suitable as a sample dilution solution.

  Alternatively, home and office use equipment for rapid processing of samples can be used. When used, the sample is placed directly into the device and testing is performed within minutes of sampling. In such cases, any solution that minimizes the need to stabilize the analyte and facilitates the assay and is not detrimental to the stability of the analyte can be used. .

Delivery Markers Any biochemical marker of imminent delivery that is assayed in cervical vaginal secretions can be used in this method. Suitable markers include, for example, elastase, total fibronectin, and fetal fibronectin. Most preferred is fetal fibronectin. Other markers for predicting imminent delivery in cervical vaginal secretion samples are known and useful in the present method.

  Elastase is an effective indicator of impending delivery in patients from about 12 weeks gestation to delivery. This marker is generally present in cervical vaginal secretion samples at a level of about 30 units of elastase per liter beginning about 2-3 weeks before parturition. Values less than 30 units per liter are considered negative.

  Total fibronectin is an effective indicator of impending delivery in patients from about 12 weeks gestation to delivery. This marker is generally present in cervical vaginal secretion samples, generally beginning about 2-3 weeks before parturition. The presence of elevated levels of total fibronectin is indicative of imminent delivery. Preferably, the total fibronectin concentration in the sample is at least about 600-750 ng / ml after a 20 week gestation period. Between 12 and 20 weeks, the threshold changes. Values below this special threshold are considered negative.

  Fetal fibronectin is an effective indicator of impending delivery in patients from about 20 weeks gestation to delivery. This marker is generally present at a level of about 50 ng / ml in cervical vaginal secretion samples beginning about 1-2 weeks before parturition.

Assay Procedures When tested, labor markers are tested by any method that measures an increased risk of labor. For example, a level of total fibronectin above a threshold is indicative of imminent delivery. However, levels of fetal fibronectin above the background (eg, the presence of fetal fibronectin) are indicative of imminent delivery.

The following discussion relates to fetal fibronectin as an example of a delivery marker, and IGFBG-1 as an example of how to measure the marker in a cervical vaginal secretion sample. IGFBP-1 is a quantitative or semi-quantitative procedure that can measure the amount of IGFBP-1 in a sample or whether the amount of IGFBP-1 exceeds a threshold amount that indicates membrane rupture. Is tested by For parturition markers, the marker is assayed by a procedure where either the amount of the marker in the sample can be measured or the amount of the marker is above a threshold indicative of imminent parturition.

  An immunoassay is preferred. The antibody affinity required to assay an analyte using a particular immunoassay method will not differ from that required to assay other polypeptide analytes. The antibody composition can be polyclonal or monoclonal. Anti-IGFBP-1 antibodies can be produced by a number of methods. Polyclonal antibodies can be derived by administering to a host animal an immunogenic composition comprising human IGFBP-1. Alternatively, a high level source of amniotic fluid or IGFBP-1 can be used as the immunogen and antibodies of the desired specificity can be identified.

  The preparation of immunogenic compositions of IGFBP-1 can vary depending on the host animal and is well known. For example, IGFBP-1 or an antigenic protein thereof can be combined with an immunogenic substance, such as KLH or BSA, and provided in an adjuvant or the like. The induced antibody can be tested to determine if the composition is IGFBP-1-specific. If a polyclonal antibody composition does not provide the desired specificity, the antibody can be purified to enhance specificity by a variety of conventional methods. For example, the composition can be purified to reduce binding to other substances by contacting the composition with IGFBP-1 immobilized on a solid support. Those antibodies that bind to the substance are retained. Purification techniques using antigens immobilized on affinity chromatography materials including various solid supports such as Sephadex, Sepharose, etc. are well known.

  Monoclonal IGFBP-1-specific antibodies can also be prepared by conventional methods. Mice can be injected with an immunogenic composition comprising IGFBP-1 and spleen cells can be obtained. These spleen cells can be fused with a fusion partner to prepare hybridomas. Screen for antibodies selected by the hybridoma to select hybridomas that show that the antibody reacts with IGFBP-1 and does not substantially react with other proteins that may be present in the sample can do. Hybridomas producing antibodies of the desired specificity are cultured by standard techniques. Hybridoma preparation techniques and culture methods are well known and do not form part of the present invention.

  Exemplary preparations of polyclonal and monoclonal are described in the examples. Antibody preparation and purification methods are described, for example, in Tijssen, P .; Laboratory Techniques in Biochemistry and Molecular Biology: Practice and Theories of Enzyme Immunoassays New York: Elsvier (1985) and are described in numerous publications and examples.

  A number of different types of immunoassays using various protocols and labels are well known. The assay conditions and reagents can be in the prior art with any variation. The assay can be a heterogeneous or homogeneous, conveniently a sandwich assay.

  The assay typically uses a solid phase-immobilized anti-IGFBP-1 antibody. The antibody can be polyclonal or monoclonal, preferably monoclonal. The solid phase-fixed antibody is merged with the sample. Binding between the antibody and the sample can be measured in a number of ways. Complex formation can be measured by the use of soluble antibodies specific for IGFBP-1. The antibody can be directly labeled or detected using a labeled second antibody specific for the soluble antibody species. Various labels include radionuclides, enzymes, fluorescent agents, colloidal metals, and the like. Conveniently, the assay will be a quantitative enzyme-linked immunosorbent assay (ELISA) such that an antibody specific for IGFBP-1 is used as a solid phase-immobilized and enzyme labeled soluble antibody. Alternatively, the assay can be based on competitive inhibition such that IGFBP-1 in the sample competes with a known amount of IGFBP-1 for a given amount of anti-IGFBP-1 antibody. For example, any of the IGFBP-1 present in the sample can compete with a known amount of labeled IGFBP-1 or IGFBP-1 analog for the antibody binding site. The amount of labeled IGFBP-1 immobilized on the solid phase or remaining in solution can be measured.

Appropriate dilution of the conjugate can be performed to detect a selected threshold level of IGFBP-1 that exceeds the background value for the assay as a positive sample.

Interpretation of test results
IGFBP-1 levels below 20-50 ng / ml are considered background and are negative. The choice of cut-off for this background level depends on whether a sensitive or highly specific test is desired. For example, as described in the Examples, a positive fetal fibronectin test (> 50 ng / ml) is shown for imminent delivery and fern, pooling and nitrazine for membrane rupture When 42 negative cervical vaginal secretion samples were tested for IGFBP-1, one of these samples showed 42 ng / ml IGFBP-1. If a 20 ng / ml cut-off was used, the suggested specificity of the test to rule out would be 97%. On the other hand, if 50 ng / ml was used, the determined specificity of the test would be 100%. In most cases, high rule-out specificity will be the preferred method because patients with membrane rupture are at higher risk of infection than those without rupture, and therefore A preferred cut-off is 20-50 ng.

  A cut-off of 20-50 ng / ml was determined for the assay described in the examples. As is well known, other assays using different reagents can have different cut-off values. For example, IGFBP-1 antibodies that differ in their antigen binding properties can produce assay results with different optimal cutoff values. One skilled in the art will recognize that background values can vary when different reagents are used, and how to determine the appropriate background level for the desired specificity and sensitivity for the selected assay. You will understand what to decide on.

  The presence of IGFBP-1 in cervical vaginal secretion samples from patients who are positive for markers of increased risk of delivery indicates that the membrane has ruptured. If IGFBP-1 is less than 20-50 ng / ml or cannot be detected (background for the assay), the membrane is intact. When IGFBP-1 is positive (> 20-50 ng / ml) and its delivery marker is negative, the amniotic membrane may be ruptured. However, most patients with ruptured membranes will show both IGFBP-1 and parturition markers, both positive at the same time.

  As previously mentioned, this test can be performed on any pregnant woman who has been tested positive for a marker indicative of an increased risk of labor. In a preferred embodiment, the delivery marker and IGFBP-1 are tested in the same cervical vaginal secretion sample. Such a test can be performed on any pregnant patient within the gestational age range for which the delivery marker is valid. Preferably, it can be done for all women with any known risk factors until postpartum after 12 weeks gestation.

  If the delivery marker is negative, the woman is not at risk for imminent delivery. This test can be repeated at regular prenatal visits throughout the gestation period or more frequently if the patient is high risk.

  If the delivery marker test is positive (above that threshold), the patient is tested for the presence of IGFBP-1 in her cervical vaginal secretions. If IGFBP-1 is present in the secretion, the patient has a ruptured membrane. If IGFBP-1 is negative, the membrane is intact.

  When IGFBP-1 is negative, the IGFBP-1 test can be repeated, preferably daily, until the sample is positive for IGFBP-1. Furthermore, if a marker is used, such as fibronectin that can be more positive in the earlier weeks than fetal fibronectin, a marker that appears closest to parturition can be used. If the parturition marker is positive and IGFBP-1 is not present in the sample, means can be devised to measure or enhance fetal lung maturation. For example, amniotic fluid samples can be analyzed for phospholipids. In general, patients at risk for preterm birth are examined every 2 weeks for about 22-36 weeks, rather than every 4 weeks for patients in the low risk category. All patients are examined about every week from about 36 weeks.

  The invention is further illustrated by the following specific but non-limiting examples. Unless otherwise specified, temperatures are given in degrees Celsius and concentrations are given as weight percentages. Procedures that are constitutively organized until implementation are described in present tense, and procedures performed in the laboratory are described in past tense.

Example 1
Quantification of fetal fibronectin in vaginal swab samples The immunoassay for measuring fetal fibronectin in vaginal samples used the reagents and procedures described below.

Preparation of monoclonal anti-fetal fibronectin antibody
The preparation of the hybrodoma deposited with the American Type Culture Collection and given the accession number ATCC HB 9018 is described in US Pat. No. 4,894,326 (incorporated by reference) to Matsuura et al on January 16, 1990. (Incorporated herein)).

  The hybridoma was cultured by growth in PRMI 1640 tissue culture medium supplemented with 10% fetal bovine serum. Furthermore, the hybridomas were cultured in mice by injection of the hybrid cells according to the procedure of Mishell and Shiigi (Selected Methids in Cellular Immunology, WH Freeman & Co, San Francisco p 368, 1980).

  A monoclonal antibody designated FDC-6 and produced by the hybridoma was prepared for use in an immunoassay by the following procedure. The IgG fraction of the culture supernatant or ascites was precipitated by ammonium sulfate fractionation, and the antibody was in a buffer suitable for purification by affinity chromatography on Protein-G Fast Flow (Pharmacia Fine Chemicals) according to the manufacturer's instructions. Redissolved and dialyzed.

Preparation of anti-fetal fibronectin antibody-coated microtiter plates Microtiter plates were coated with FDC-6 monoclonal antibody by the procedure described below.

  Monoclonal antibody FDC-6, prepared as described above, was diluted to 10 μg / ml in phosphate buffer, pH 7.2, and 100 μl / well was dispersed in polystyrene microtiter plates (Costar). Plates were incubated for 2 hours at room temperature or overnight at 4 ° C. The contents of the wells were aspirated and the wells were washed 3-4 times with wash buffer (0.02 M Tris HCl, 0.15 M NaCl, 0.05% TWEEN-20). 200 μl / well blocking / stabilizing solution (4% sucrose, 1% mannitol, 0.5% casein, 0.01 M PBS) was then added to the wells and incubated at room temperature for 30 minutes to 4 hours. The wells were then evaporated to dryness and the plate was packaged in an airtight container with a desiccant pouch.

  The above procedure was repeated using microtiter plates from Nunc and Dynatech and obtained equivalent results.

Preparation of enzyme-labeled anti- (fibronectin) antibody Human plasma fibronectin was prepared as described in Engvall and Ruoslahti, Int. J. Purified from human plasma as described by Cancer 20: 1-5 (1977). This anti-human plasma fibronectin antibody has been described in the literature, eg Stollar, Meth. Enzym. The immunization techniques and schedule described in 70:70 (1980) were used to elicit immunization of the goat with this human plasma fibronectin antigen. This antiserum is described, for example, in Lange et al, Clin. Exp. Immunol. 25: 191 (1976) and Pisetsky et al. Immun. Meth. 41: 187 (1981) screened in a solid phase assay similar to that used for monoclonal antibodies.

  Using the IgG fraction of this antiserum recommended by the manufacturer (AFFINITY CHOROMATOGRAPHY, Pharmacia Fine Chemicals Catalog 1990), CNBr-Sepharose 4B (Pharmacia Fine Chemicals) conjugated with human plasma fibronectin according to pages 15-18 Further purification by affinity chromatography.

  Briefly, the column was equilibrated with 2-3 volumes of buffer (0.01 MPBS, pH 7.2) and an anti-human fibronectin antibody-containing solution was then applied to the column. The absorption of this eluate was monitored at 280 nm until protein no longer passed through the column. The column was then washed with equilibration buffer until a baseline absorbance at 280 nm was obtained.

  Immunoaffinity bound anti-human plasma fibronectin antibody was eluted with 0.1 M glycine buffer, pH 2.5. Peak protein fractions were collected, pooled, and dialyzed against 0.01 M PBS, pH 7.2 at 4 ° C for 24-36 hours with multiple buffer changes.

  The procedure above was repeated to immunize rabbits with human plasma fibronectin and purify the resulting polyclonal anti-human fibronectin antibody.

  Anti-human plasma fibronectin prepared as described above was prepared according to Avrameas, Immunochem. Conjugated with alkaline phosphatase following the one-step glutaraldehyde procedure of 6:43 (1969).

Assay Reagents Assays were performed using the following additional reagents. Conserved antibody conjugate is conjugate diluent (0.05 M Tris Buffer pH 7.2, 2% D-sorbitol, 2% BSA, 0.1% sodium azide, 0.01% Tween-20, 1 mM magnesium chloride, and 0.1% zinc chloride) Dilute appropriately in and 10 ml was placed in a polyethylene dropper bottle container.

  Enzyme substrate (10 ml in polyethylene dropper bottle container) is phenolphthalein monophosphate dissolved in 0.4 M aminomethylpropanediol buffer, pH 10 containing 0.1 mM magnesium chloride and 0.2% sodium azide ( 1 mg / ml).

  The positive control (2.5 ml in polyethylene dropper bottle container) was sample diluted solution (0.05 M Tris buffer pH 7.4, 1% BSA, 0.15 M sodium chloride, 0.02% sodium azide, 5 mM ethylenediaminetetraacetic acid (EDTA), Amniotic fluid containing fetal fibronectin diluted to a concentration of 50 ng / ml fetal fibronectin in 1 mM phenylmethylsulfonyl fluoride (PMSF) and 500 Kallikrein Units / ml aprotinin). This sample dilution is described in US Pat. No. 4,919,889, issued April 24, 1990 to Jones et al, which is incorporated herein by reference in its entirety.

  The negative control (2.5 ml in a polyethylene dropper bottle container) was the sample dilution solution used for the positive control without fetal fibronectin.

  The rinse buffer (10 ml in a polyethylene dropper bottle container) was a 50-fold concentrate containing 1.0 M Tris buffer pH 7.4, 4.0 M sodium chloride, 2.5% Tween-20, and 1% sodium azide. The rinse buffer was diluted with water to a final concentration of 0.02 M Tris, 0.08 M sodium chloride, 0.05% Tween-20, and 0.02% sodium azide for use in this assay.

  In addition, a 5μ pore size polyethylene sample filter (Porex Technologies, Fairburn, Georgia) was used to filter the sample prior to assay. All of the dropper bottles used to perform this assay were polyethylene bottles designed to dispense approximately 50 μl drops of reagent. All of the assay steps performed after sample collection used the reagents and materials described above.

Test procedure The test was performed as follows. All samples were collected using a Dacron swab near the posterior fornix and in the cervical os. A swab sample was immersed in a 1.0 ml sample dilution in a collection vial. A swab was removed from the solution, leaving as much of the liquid as possible in the collection tube. Samples were incubated at 37 ° C. with controls for 15 minutes prior to the assay, either before or after filtration. The sample filter was snapped to a location above each sample tube. Each sample and duplicate 100 μl aliquots of positive and negative controls were placed in separate wells of a microtiter plate and incubated for 1 hour at room temperature.

  Following incubation, samples and controls were aspirated from the wells. Wells were washed 3 times with diluted wash buffer (1x). After washing, 100 μl of enzyme-antibody conjugate was added to each well and incubated for 30 minutes at room temperature. Wells were aspirated and washed as previously described. After washing, 100 μl of enzyme substrate was added to each well and incubated for 30 minutes at room temperature.

  After the incubation, the plate was gently agitated by hand or with an elliptical shaker to mix the well contents. The strip frame was placed in an ELISA plate reader. The absorbance of each well at 550 nm was measured. Absorbance of duplicate wells for each sample and control was calculated. The fetal fibronectin concentration for the sample was prepared as a standard curve and the sample was diluted to about 1/10 of their original concentration (collection of about 0.1 ml sample combined with 1.0 ml dilution) It was calculated by estimating that. For this study, a cut-off of approximately 50 ng / ml was used as a positive sample. Samples below 50 ng / ml were considered background and negative.

Example 2
Detection of IGFBP-1 in vaginal swab samples
IGFBP-1 was detected by the procedure described below.

Preparation of anti-IGFBP-1 monoclonal antibody A panel of hybridomas was generated by immunization of mice with human amniotic fluid. One monoclonal antibody (designated AF127) reacted with a 31 kd protein that was found to be one of the most abundant proteins in amniotic fluid. A two-dimensional gel western blot was used to identify this polypeptide antigen. Since this protein is very abundant in baboon amniotic fluid, it was transferred to a polyvinylidene difluoride membrane, a suitable absorber for sequencing the protein.

  The N-terminus of the protein is described in Applied Biosystems, Inc. The Model 477A amino acid sequencer was used to sequence using a single paper disk punched with a conventional paper hole puncher containing 100 picomoles of protein amino acids with an Edman degradation amino acid sequencer. This N-terminal sequence was determined to be APWQCAPCSAEKLALCPPVPASCSEVTRSA (SEQ ID NO: 1), which identified the protein as IGFBP-1 using GenBank.

  A monoclonal antibody designated AF127 and produced by a hybridoma was prepared for use in an immunoassay by the following procedure. The IgG fraction of the culture supernatant or ascites was purified using Avid Al affinity gel purification for immunoglobulins according to the manufacturer's instructions (Bioprobe International, Inc. Tustin, CA).

Preparation of anti-IGFB0P-1-coated microtiter plates Microtiter plates were coated with IGFBP-1 monoclonal antibody by the procedure described below.

The monoclonal antibody IGFBP-1 prepared as described above is diluted to 10 μg / ml in PBS (0.01 M phosphate buffer, 0.15 M NaCl, pH 7.4, 0.02% NaN ₃ ), and 100 μl / well is diluted with polystyrene. -Dispersed in a microtiter plate (Costar). Plates were incubated overnight at 4 ° C. The contents of the wells were aspirated and washed once with wash buffer (0.02 M Tris HCl, pH 7.9, 0.15 M NaCl). 250 μl / well blocking solution (3% IGFBP-1-free BSA in PBS) was then added to the wells and incubated for 2 hours at room temperature. Wells were aspirated and then washed once and stored as described above.

Preparation of polyclonal anti-IGFBP-1 antibody
IGFBP-1 was purified from baboon amniotic fluid using gel electrophoresis followed by electroelution / electrotransfer. Anti-baboon IGFBP-1 antibodies were developed in goats using standard immunization techniques and schedules (by immunizing goats with baboon amniotic fluid containing IGFBP-1.

  This antiserum is described, for example, in Lange et al, Clin. Exp. Immunol. 25: 191 (1976) and Pisetsky et al. Immmun. Meth. 41: 187 (1981) screened in a solid phase assay similar to that used for monoclonal antibodies.

Assay Reagents Assays were performed using the following additional reagents.

Commercially available swine anti-goat alkaline phosphatase antibody conjugate (TAGO, Burlingame, California) and conjugate dilution (0.02 M Tris buffer, pH 7.9, 1% BSA, 0.1% sodium azide) , 0.05% TWEEN-20). The enzyme substrate was phenolphthalein monophosphate (1 mg / ml) dissolved in 0.4 M aminomethylpropanediol buffer, pH 10, containing 0.1 mM MgCl ₂ and 0.2% sodium azide.

  The positive control is human amniotic fluid diluted to a concentration of 50 ng / ml IGFBP-1 in sample dilution solution (0.02 M Tris buffer, pH 7.9, 0.5% BSA, 0.15% sodium chloride, 0.02% sodium azide). there were.

  The negative control was the sample dilution solution used for the positive control without IGFBP-1.

IGFBP-1 Assay Procedure Cervical vaginal secretion samples were prepared as described in Example 1. Each sample or dilution thereof and duplicate 100 μl aliquots of positive and negative controls were placed in separate wells of a microtiter plate and incubated for 2 hours at room temperature.

  After incubation, the wells were washed 3 times in rinse buffer (0.02 M Tris, pH 7.0, 0.15 M NaCl, 0.05% TWEEN-20, and 0.02% sodium azide).

  After rinsing, 100 μl goat anti-IGFBP-1 antibody (1: 200 dilution) was added to each well and incubated for 2 hours at room temperature. After incubation, the plate was washed 3 times in rinse buffer. After rinsing, 100 μl of porcine anti-goat conjugate (1: 4,000 dilution) was added to each well and incubated for 1 hour at room temperature. Following incubation, the plate was rinsed once in buffer and 100 μl of enzyme substrate was read immediately at 404 nm using an ELISA plate reader. The plate was read again after 30 minutes and the end point reading was taken.

  The average absorbance of duplicate wells for each sample and control was calculated. The IGFBP-1 concentration for the sample was calculated by preparing a standard curve using amniotic fluid containing a known concentration of IGFBP-1.

Example 3
Patient Panel Study A panel of cervical secretion samples from second and third trimester patients were tested for fetal fibronectin as described in Example 1. This panel was tested for membrane rupture using conventional fern, pooling, and nitrazine. The same panel was then tested for IGFBP-1.

  IGFBP-1 could not be detected at less than 20-40 ng / ml (<10 ng / ml) in samples that were negative for fern crystal formation, pooling, and membrane disruption by nitrazine. In addition, IGFBP-1 is either fetal fibronectin positive (> 50 ng / ml), negative membrane rupture (due to fern crystal formation, pooling, and nitrazine) and either premature birth positive or negative as determined by results ( Whether the patient was delivered during or before the 37-week gestation period) was negative in samples from women. Most patients who were positive for fern crystal formation, pooling, and membrane disruption by nitrazine were also positive for IGFBP-1 (range 30-> 5000 ng / ml).

  Circulating levels of IGFBP-1 in maternal plasma were examined to determine if blood contamination of cervical vaginal secretions would interfere with testing for IGFBP-1. The levels of IGFBP-1 in maternal plasma ranged from less than 10 ng / ml to 250 ng / ml and averaged about 150 ng / ml. Most membrane rupture positive cervical vaginal secretion samples recorded levels of IGFBP-1 greater than 250 ng / ml. Therefore, the level of IGFBP-1 in the cervical vaginal secretion is determined by the rupture of membrane (ROM) in the presence or absence of 10% blood in the cervical vaginal secretion sample. Is a reliable indicator. Moreover, when fetal fibronectin is positive (> 50 ng / ml), the absence of IGFBP-1 is a reliable indicator that membrane rupture did not occur even in the presence of fetal fibronectin. The ability to rule-out membrane rupture assists physicians in determining approaches to clinical management of pregnancy.

Example 4
Patient Panel Study In a second panel of patients, 4 groups of pregnant women were tested for fetal fibronectin and IGFBP-1 in cervical secretions. These groups were: (Group 1) positive for preterm birth (PTD +; delivery before 37 weeks) and fetal fibronectin positive (fFN +;> 50 ng / ml) but negative for fern crystal formation, pooling, and membrane disruption by nitrazine Patients with (ROM-); (Group 2) PTD- (delivery after 37 weeks) but fFN + (> 50 ng / ml) and R0M- with fern crystal formation, pooling, and nitrazine (Group 3) Patients who are PTD- (delivery after 37 weeks) and fFN- (<50 ng / ml) and R0M- with fern crystal formation, pooling, and nitrazine; Group 4) patients with positive fertility, pooling, and nitrazine-induced membrane rupture (ROM +), regardless of gestation period.

  In one group, 24 or 23 cervical secretion samples showed less than 20 ng / ml of IGFBP-1, while one sample from a patient sampled at 27 weeks gestation was ml 42 ng / ml IGFBP-1 was shown in samples that also showed more than 1 microgram of fetal fibronectin per minute. Therefore, if the cut-off of the membrane rupture (ROM) determination test was 50 ng IGFBP-1 per ml, its determination specificity (ROM determination specificity is the number of IGFBP-1 negative compared to true ROM negative Would be 100% based on fern crystal formation, pooling, and ROM diagnosis with nitrazine. If the cut-off was less than 40 ng IGFBP-1 per ml, the ROM determination specificity would be 96%.

  In group 2, 27 out of 27 cervical secretion samples showed less than 20 ng / ml IGFBP-1. Thus, in this group, ROM determination was 100% specific based on ROM diagnosis by fern crystal formation, pooling, and nitrazine test.

  In group 3, 23 out of 23 cervical secretion samples showed less than 20 ng / ml IGFBP-1. Thus, in this group, ROM determination was again 100% specific based on ROM diagnosis by fern crystal formation, pooling, and nitrazine test.

  In group 4, 24 out of 30 samples showed more than 20 ng / ml IGFBP-1. Thus, in this group of patients diagnosed with ROM + based on fern crystal formation, pooling, and nitrazine tests, the IGFBP-1 test was 80% specific in ROM diagnosis. However, one of the six IGFBP-1 negative patients showed a negative fetal fibronectin test (<50 ng / ml). This should have been positive when amniotic fluid was present. This is because fetal fibronectin exists in amniotic fluid.

  The discovery that two abundant markers in amniotic fluid (IGFBP-1 and fetal fibronectin) gave results that were inconsistent with the results of the more subjective criteria of fern crystal formation, pooling, and nitrazine results The problem is the reliability of fern crystal formation, pooling, and nitrazine testing for accurate diagnosis of rupture. It is well known that fern crystal formation, pooling, and nitrazine are a combination of tests that are unsuitable for measuring bursts. In particular, amniotic fluid appears to be present when the test result is positive. However, a negative result cannot indicate that there is no amniotic fluid. This is because the sensitivity of the test is low. However, since this test is subjective, both positive and negative results cannot be accurate.

Claims (11)

An immunoassay method that distinguishes intact and ruptured fetal membranes in pregnant patients who have been determined to be at risk of imminent preterm birth:
a) Imminence by detection of biochemical markers for imminent delivery selected from the group consisting of elastase, total fibronectin and fetal fibronectin in cervical vaginal secretions from pregnant patients at 12 weeks of gestation or later Determining which pregnant patients are at risk of preterm birth; and
b) Insulin-like growth factor binding protein 1 levels in cervical vaginal samples are measured from among patients diagnosed as at risk of imminent premature delivery, and insulin- exceeding the threshold level in the sample The presence of the like-like growth factor binding protein 1 indicates rupture of the fetal membrane of a pregnant patient who has been determined to be at risk of imminent preterm birth , where the threshold level of insulin-like growth factor binding protein 1 is the back of the immunoassay Comprising a level above the ground value ,
Immunoassay method. The method of claim 1, wherein the sample is a posterior vaginal cap sample . The method of claim 1, wherein the sample is a cervical mouth sample .   2. The method of claim 1, wherein the biochemical marker for imminent delivery is fetal fibronectin.   2. The method of claim 1, wherein fetal fibronectin and insulin-like growth factor binding protein 1 are tested on the same sample.   2. The sample of claim 1, wherein the sample does not contain elevated levels of insulin-like growth factor binding protein 1 and the other sample from the patient is assayed for insulin-like growth factor binding protein 1 after at least one day. the method of.   Samples from the patient were obtained at 20-week gestation periods, at 2-week intervals until the patient reached the period or elevated insulin-like growth factor binding protein 1 levels were measured, and fetal fibronectin And the method of claim 1, wherein the method is assayed for the presence of insulin-like growth factor binding protein 1. A method to determine imminent labor and membrane rupture in pregnant patients after a 20-week gestation period:
To determine the pregnant patients at risk of a) impending delivery, patients vaginal cavity or after gestation period of 20 weeks and tested secretion sample from the cervix, the fetus above a threshold level in the sample Detecting the presence of fibronectin ; and
in a sample obtained from a pregnant patient that is determined to be at risk of b) impending delivery, insulin in the sample - determining the presence above a threshold level of like growth factor binding protein 1, contains become ,
The presence of fetal fibronectin above the threshold level in the sample indicates imminent delivery ;
In the impending delivery patients, insulin exceeds the threshold level in the sample - the absence of like growth factor binding protein 1, shows the intact fetal membranes; and
The threshold level is above the background value of the immunoassay;
Method. A method for determining whether a pregnant patient after a 20 week gestation period has imminent labor and an intact membrane:
To determine the pregnant patients at risk of a) impending delivery, patients vaginal cavity or after gestation period of 20 weeks and tested secretion samples from cervical, elastase in the sample, total fibronectin, And measuring the level of imminent delivery markers selected from the group consisting of fetal fibronectin ;
in a sample obtained from a pregnant patient that is determined to be at risk of b) impending delivery, insulin in the sample - determining the presence above a threshold level of like growth factor binding protein 1, contains become ,
The presence of an impending delivery marker above the threshold level in the sample indicates imminent delivery ;
In the impending delivery patient, the insulin below a threshold level in the sample - like the presence of growth factor binding protein 1, shows the intact fetal membranes; and
The threshold level is above the background value of the immunoassay;
Method.   An immunoassay kit comprising an anti-insulin-like growth factor binding protein 1 antibody and an antibody specific for an imminent delivery marker selected from the group consisting of elastase, total fibronectin, and fetal fibronectin.   The immunoassay kit according to claim 10, wherein the imminent delivery marker is fetal fibronectin.