METHOD FOR DETECTING IMMINENT CHILDBIRTH
20220057407 · 2022-02-24
Inventors
Cpc classification
G01N2800/60
PHYSICS
G01N33/74
PHYSICS
G01N2800/368
PHYSICS
International classification
Abstract
Some embodiments are directed to a method for the in vitro detection/prediction of imminent childbirth based on the detection of at least two of the following markers: insulin-like growth factor-binding protein 1 (IGFBP-1), a N-terminal fragment of insulin-like growth factor-binding protein 1, and interleukin 6 (IL-6). Some embodiments are also directed to a device for the implementations of the method. Some embodiments are applicable in the medical field, in the diagnostic field, and in particular in the obstetric field.
Claims
1. A method for the in vitro detection/prediction of an imminent birth, comprising: simultaneously searching, in a specimen of vaginal or cervical secretions, for markers of a the group consisting of intact insulin-like growth factor binding protein 1 (IGFBP-1 A), of intact insulin-like growth factor binding protein 1 and/or an N-terminal fragment of insulin-like growth factor binding protein 1 (IGFBP-1 B) and of interleukin 6 (IL-6); and detecting or predicting an imminent birth if at least two of the markers of the group consisting of insulin-like growth factor binding protein 1 A (IGFBP-1 A), of insulin-like growth factor binding protein 1 B (IGFBP-1 B) and of interleukin 6 (IL-6), are detected.
2. A method for the in vitro detection/prediction of an imminent birth in a sample of vaginal or cervical secretions, comprising: a) searching for the presence of IGFBP-1 A, of IGFBP-1 B and of IL-6, b) assigning a value of IGFBP-1 A (I.sub.A) if IGFBP-1 A is present in the sample or of 0 if IGFBP-1 A is not present, c) assigning a value of IGFBP-1 B (I.sub.B) if IGFBP-1 B is present in the sample or of 0 if IGFBP-1 B is not present, the value of I.sub.B assigned if IGFBP-1 B is present in the sample being less than or equal to the value of I.sub.A assigned when IGFBP-1 A is present in the sample, d) assigning a value of IL-6 (I.sub.6) if IL-6 is present in the sample or of 0 if IL-6 is not present, the value I.sub.6 assigned when IL-6 is present in the sample being strictly less than the values of I.sub.A and I.sub.B assigned when IGFBP-1 A and IGFBP-1 B are respectively present in the sample, e) calculating the score S1 according to the following formula:
S.sub.1=I.sub.A+I.sub.B+I.sub.6 a value of S1 greater than or equal to the value of I.sub.A or I.sub.B, respectively assigned when IGFBP-1 A or IGFBP-1 B is present in the sample, indicating an imminent birth.
3. The method as claimed in claim 1, wherein the searching is carried out with at least one capture antibody directed against IL-6, at least one capture antibody directed against IGFBP-1 A and at least one capture antibody directed against IGFBP-1 B.
4. The method as claimed in claim 2, wherein the searching is carried out using a sample of vaginal or cervical secretions diluted in a buffer solution.
5. The method as claimed in claim 1, wherein the searching is carried out using a specimen of vaginal or cervical secretions diluted in a buffer solution.
6. The method as claimed in in claim 2, further comprising at least one of the parameters chosen from the volume, the pH and the ionic strength of the buffer medium.
7. The method as claimed in claim 5, further comprising adjusting at least one of the parameters chosen from the concentration of the capture antibodies, the adjustment being specific to IL-6, to IGFBP-1 A or to IGFBP-1 B.
8. The method as claimed in claim 5, further comprising adjusting at least one of the parameters chosen from the concentration of the capture antibodies, the adjustment being specific to IL-6, to IGFBP-1 A and/or to IGFBP-1 B.
9. The method as claimed in claim 5, wherein the pH of the buffer solution is from 5 to 10.
10. The method as claimed in claim 1, wherein the searching step includes a revealing step carried out with at least one labeled anti-IGFBP-1 A antibody, at least one labeled anti-IGFBP-1 B antibody and at least one labeled anti-IL-6 antibody.
11. The method as claimed in claim 1, wherein the IGFBP-1 A is detected starting from a concentration of 40 ng/ml.
12. The method as claimed in claim 1, wherein the IGFBP-1 B is detected starting from a concentration of 20 ng/ml intact and/or 10 ng/ml of the N-Ter fragment of IGFBP-1.
13. The method as claimed in claim 1, wherein the IL-6 is detected starting from a concentration of 0.25 ng/ml.
14. A device for implementing the method as claimed in claim 1, comprising: a sample-depositing zone 1, a zone 2 including labeled anti-IGFBP-1 A antibodies, labeled anti-IGFBP-1 B antibodies and labeled anti-IL-6 antibodies, a revealing zone 3 including capture antibodies directed against IGFBP-1 A, a revealing zone 4 including capture antibodies directed against IGFPB-1 B, a revealing zone 5 including capture antibodies directed against IL-6.
15. The device as claimed in claim 14, wherein the depositing zone 1 is chosen from the group including an absorbent paper and a glass fiber support.
16. The device as claimed in claim 14, wherein the revealing zone 4 and/or 5 is a nitrocellulose membrane.
17. The device as claimed in claim 14, wherein the zone 2 and/or 3 is a glass fiber support.
18. The device as claimed in claim 14, wherein the zones 1, 2, and/or 3 are a glass-fiber or nitrocellulose support.
19. (canceled)
20. The method as claimed in claim 2, wherein the searching is carried out with at least one capture antibody directed against IL-6, at least one capture antibody directed against IGFBP-1 A and at least one capture antibody against IGFBP-1 B.
21. The method as claimed in claim 3, wherein the searching is carried out using a sample of vaginal or cervical secretions diluted in a buffer solution.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0156]
[0157]
[0158]
[0159]
[0160]
[0161]
[0162]
EXAMPLES
Example 1: Immunochromatographic Device for Evaluating the Risk of Imminent Birth
[0163] The device is composed of three distinct strips: a strip detecting IGFBP-1 A, a strip detecting IGFBP-1 B and a strip detecting IL-6.
Preparation of the IL-6 Strip
[0164] The strips were prepared from laminated cards of 30 cm×6 cm (CNPC-SS12, MDI (registered trademark)) including or consisting of a plastic support covered with a layer of adhesive on which the nitrocellulose membrane, the absorbent paper and the glass fibers are assembled.
[0165] Using an automated reagent dispenser (Isoflow Dispenser, Imagene Technology Inc. (registered trade mark)), goat anti-mouse IgG antibodies (ABGAM-0500, Arista Biologicals (registered trade mark)) diluted to 0.5 mg/ml in a PBS buffer and also anti-IL-6 antibodies (catalog number 855.050.005, clone B-E8, Diaclone (registered trade mark)) diluted to 1 mg/ml in a PBS buffer were deposited, with a flow rate of 1 μl/cm, in the form of parallel lines 5 mm apart and having a width of 1 to 2 min each, on a nitrocellulose membrane, the width of which is 25 mm.
[0166] After deposition of these searching methods, these cards were then placed for 30 minutes in an oven at 37° C. under an atmosphere with a controlled humidity of less than 30% in order to dry them.
[0167] The conjugate mixture, that is to say a solution including labeled antibodies, was prepared from the anti-IL-6 antibodies (catalog number 879.030.002, clone B-E4, Diaclone (registered trademark)) coupled beforehand to colloidal gold particles according to the following protocol:
[0168] 1.0 ml of 1% gold chloride solution (G4022, Sigma Aldrich) was added to 100 ml of distilled water. The water was heated to boiling point and 2.5 ml of 1% sodium citrate solution (S1804, Sigma Aldrich) were added. The solution became colorless and then turned purple. After one minute, the solution was heated until it became cherry red. 9.5 ml of 1% sodium citrate (S1804, Sigma Aldrich) were added. The water was heated to boiling point and 8.6 ml of 1% gold chloride solution (G4022, Sigma Aldrich) were added. The solution became dark blue, virtually black, then purple, and then was left to cool at ambient temperature.
[0169] The pH of 10 ml of the previously obtained purple colloidal gold solution was adjusted, using a 0.2 M potassium carbonate solution (269619, Sigma Aldrich), until a pH of 6.3 was reached. 200 μg of anti-IL-6 were added to 1 ml of distilled water and then 10 ml of purple colloidal gold solution with the pH adjusted to 6.3 were rapidly added. This tube was placed on a circular mixer (multidisk rotator 5-50 rpm, Sobinco (registered trademark)) for 20 minutes. 1.5 ml of Gold stabilization Buffer (Artron Bioresearch (registered trademark)) were added. The 2 tubes were centrifuged (5804, Eppendorf (registered trademark)) at 4000 ref for 30 minutes. The supernatant was suctioned off using a vacuum pump (159600, Brand (registered trademark)) and the pellet was taken up with 1 ml of resuspension buffer (pH 5.0) containing 20 mM Tris Base (26-128-3094, Euromedex (registered trademark)), 50 mM NaCl (S7653, Sigma Aldrich (registered trademark)), 0.2% BSA (1035-70, ID Bio (registered trademark)), 10% sucrose (S8501, Sigma Aldrich), 5% trehalose (T9531, Sigma Aldrich (registered trademark)).
[0170] 800 μl of the saturating solution ((pH. 74) 50 mM Na.sub.2HPO.sub.4 (S7907, Sigma Aldrich (registered trademark)), 1% of bovine serum albumin (BSA) (1035-70, ID Bio), 20% of sucrose (S8501, Sigma Aldrich) and 5% of trehalose (T9531, Sigma Aldrich (registered trademark))) were added to 200 μl of IL-6 conjugate solution. The resulting mixture was stirred for 20 minutes at ambient temperature on the circular mixer adapted for 1.5 ml tubes.
[0171] The mixture of antibodies coupled to the colloidal gold particles was then sprayed with a flow rate of 4 μl/cm onto glass fiber sheets (conjugate pad zone) having the dimensions 30 cm×1 cm (#6613 Ahlstrom, MAPDS-200, Arista Biologicals (registered trademark)) pretreated with a solution of 0.5% of polyvinyl alcohol P8136, Sigma Aldrich (registered trademark)), 0.1% of triton X-100 (X-100, Sigma Aldrich (registered trademark)), 0.5% of bovine serum albumin (BSA) (1035-70, ID Bio), 50 mM Na.sub.2HPO.sub.4 (S7907, Sigma Aldrich (registered trademark)), pH 7,4.
[0172] The binding of this mixture of conjugates to the glass fiber sheet was carried out with the same Isoflow Dispenser (registered trademark) apparatus by complying with the manufacturer's indications. After spraying, the glass fiber sheets are dried for 30 minutes in an oven at 37° C. under an atmosphere at a controlled humidity of less than 30% in order to dry them.
[0173] The cards were assembled by adhesively bonding the conjugate-impregnated glass fiber onto the lower adhesive part of the card, making the glass fiber sheet overlap by 1 to 3 mm on the nitrocellulose membrane. A second glass fiber paper (sample pad zone) having dimensions of 30 cm×1.6 cm (#8964 Ahlstrom, MAPDS-0300, Arista Biologicals (registered trademark)) pretreated with a solution of 0.1 M of B.sub.4Na.sub.2O.sub.7 (B3545, Sigma Aldrich (registered trademark)), 1% triton X-100 (X-100, Sigma Aldrich (registered trademark)), pH 8.4, is adhesively bonded in the lower adhesive part of the card while overlapping the conjugate-impregnated glass fiber sheet by 1 to 3 mm.
[0174] In the same way, a paper with high absorption capacity (absorbent pad zone) (absorbent pad, Ahlstrom 222, MAPDS-0100, Arista Biologicals (registered trademark)) having dimensions of 30 cm×1.6 cm was placed on the upper part of the card and overlaps the nitrocellulose membrane by 1 to 3 mm in order to be able to create a migration flow.
[0175] The card thus assembled was cut up into strips of 4 mm wide using a guillotine (model CM4000, Biodot, (registered trademark)).
[0176] The strips were then placed in a plastic support called a cassette (MK001, D2 Technologies (registered trademark)).
[0177] The cassettes were then closed using a clipping device (Closure-I, A-Point Technologies (registered trademark)).
[0178]
Preparation of the IGFBP-1 A Strip
[0179] The strips were prepared from laminated cards of 30 cm×6 cm (CNPC-SS12, MDI (registered trademark)) including or consisting of a plastic support covered with a layer of adhesive on which the nitrocellulose membrane, the absorbent paper and the glass fiber were assembled.
[0180] Using an automated reagent dispenser (Isoflow Dispenser, Imogene Technology Inc. (registered trademark)), goat anti-mouse IgG antibodies (ABGAM-0500, Arista Biologicals (registered trademark)) diluted to 0.5 mg/ml in a PBS buffer and also anti-IGFBP-1 antibodies (catalog number 4IG8, clone C7B9, Hytest (registered trademark)) diluted to 1 mg/ml in a PBS buffer were deposited, with a flow rate of 1 μl/cm, in the form of parallel lines 5 mm apart and from 1 to 2 mm wide each, on a nitrocellulose membrane, the width of which is 25 mm.
[0181] After deposition of these searching methods, these cards were then placed for 30 minutes in an oven at 37° C. under an atmosphere at a controlled humidity of less than 30% in order to dry them.
[0182] The conjugate mixture, that is to say a solution including labeled antibodies, was prepared from the anti-IGFBP-1 antibody (catalog number 4IG8, clone G5F8, Hytest (registered trademark)) coupled beforehand to colloidal gold particles according to the following protocol:
[0183] 1.0 ml of 1% gold chloride solution (G4022, Sigma Aldrich) was added to 100 ml of distilled water. The water was heated to boiling point and 2.5 ml of 1% sodium citrate solution (S1804, Sigma Aldrich) were added. The solution became colorless and then turned purple. After one minute, the solution was heated until it became cherry red. 9.5 ml of 1% sodium citrate. (S1804, Sigma Aldrich) were added. The water was heated to boiling point and 8.6 ml of 1% gold chloride solution (G4022, Sigma Aldrich) were added. The solution became dark blue, virtually black, then purple and was then left to cool to ambient temperature.
[0184] The pH of 10 ml of the purple colloidal gold solution previously obtained was adjusted, using a 0.2 M potassium carbonate solution (269619, Sigma Aldrich), until a pH of 7.8 was reached. 200 μg of anti-AIGFBP-1 clone F8 were added to 1 ml of distilled water and then 10 ml of purple colloidal gold solution at the pH adjusted to 7.8 were rapidly added. These tubes were placed on a circular mixer (multidisk rotator 5-50 rpm, Sobinco (registered trademark)) for 20 minutes. 1.5 ml of gold stabilization buffer (Artron Bioresearch (registered trademark)) were added. The 2 tubes were centrifuged (5804, Eppendorf (registered trademark)) at 4000 rcf for 30 minutes. The supernatant was suctioned off using a vacuum pump (159600, Brand (registered trademark)) and the pellet was taken up with 1 ml of resuspension buffer (pH 8.0) containing 20 mM Tris Base (26-128-3094, Euromedex (registered trademark)), 50 mM NaCl (S7653, Sigma Aldrich (registered trademark)), 0.2°BSA (1035-70, ID Bio (registered trademark)), 10% sucrose (S8501, Sigma Aldrich), 5% trehalose (T9531, Sigma Aldrich (registered trademark)).
[0185] 800 μl of the saturating solution ((pH 7.4) 50 mM Na.sub.2HPO.sub.4 (S7907, Sigma Aldrich (registered trademark)), 1% of bovine serum albumin (BSA) (1035-70, ID Bio), 20% of sucrose (S8501, Sigma Aldrich) and 5% of trehalose (T9531, Sigma Aldrich (registered trademark))) was added to 200.Math.1 of solution of IGFBP-1 conjugate, clone F8. The mixture thus obtained was stirred for 20 minutes at ambient temperature on the circular mixer adapted for 1.5 ml tubes.
[0186] The mixture of antibodies coupled to the colloidal gold particles was then sprayed, with a flow rate of 4 μl/cm, onto glass fiber sheets (sample/conjugate pad zone) having dimensions of 30 cm×3.2 cm (#8964 Ahlstrom, MAPDS-0300, Arista Biologicals (registered trademark)) pretreated with a solution of 0.1 M B.sub.4Na.sub.2O.sub.7 (B3545, Sigma Aldrich (registered trademark)), 1% triton X-100 (X-100, Sigma Aldrich (registered trademark)), pH 8.4.
[0187] The binding of this conjugate mixture to the glass fiber sheet was carried out with the same Isoflow Dispenser (registered trademark) apparatus while complying with the manufacturer's indications. After spraying, the glass fiber sheets were dried for 30 minutes in an oven at 37° C. under an atmosphere with a controlled humidity of less than 30% in order to dry them.
[0188] The cards were assembled by adhesively bonding the conjugate-impregnated glass fiber onto the lower adhesive part of the card while causing the glass fiber sheet to overlap by 1 to 3 mm on the nitrocellulose membrane. In the same way, a paper with a high absorption capacity (absorbent pad zone) (Ahlstrom 222, MAPDS-0100, Arista Biologicals) having dimensions of 30 cm×1.6 cm was placed on the upper part of the card and overlaps the nitrocellulose membrane by 1 to 3 mm in order to be able to create a migration flow.
[0189] The card thus assembled was cut into strips 4 mm wide using a guillotine (model CM4000, Biodot).
[0190] The strips were then placed in a plastic support called a cassette (MK001, D2 Technologies (registered trademark))
[0191] The cassettes were then closed using a clipping device (Closure-I, A-Point Technologies (registered trademark)).
[0192]
Preparation of the IGFBP-1 B Strip
[0193] The strips were prepared from laminated cards of 30 cm×6 cm (CNPC-SS12, MDI (registered trademark)) including or consisting of a plastic support covered with a layer of adhesive on which the nitrocellulose membrane, the absorbent paper and the glass fiber were assembled.
[0194] Using an automated reagent dispenser (Isoflow Dispenser, Imagene Technology Inc. (registered trademark)), goat anti-mouse IgG antibodies (ABGAM-0500, Arista Biologicals (registered trademark)) diluted to 0.5 mg/ml in a PBS buffer and also anti-IGFBP-1 antibodies (catalog number 4IG8, clone C7B9, Hytest (registered trademark)) diluted to 0.5 mg/ml in a PBS buffer were deposited, with a flow rate of 1 μl/cm, in the form of parallel lines 5 mm apart and from 1 to 2 mm wide each, on a nitrocellulose membrane, the width of which is 25 mm.
[0195] After deposition of these searching methods, these cards were then placed for 30 minutes in an oven at 37° C. under an atmosphere at a controlled humidity of less than 30% in order to dry them.
[0196] The conjugate mixture, that is to say a solution including labeled antibodies, was prepared from the anti-IGFBP-1 antibodies (catalog number 4IG8, clone G5F8, Hytest (registered trademark)) and the anti-IGFBP-1 antibodies (catalog number 4I52, clone G2, Hytest (registered trademark)) coupled beforehand to colloidal gold particles according to the following protocol:
[0197] 1.0 ml of 1% gold chloride solution (G4022, Sigma Aldrich) was added to 100 ml of distilled water. The water was heated to boiling point and 2.5 ml of 1% sodium citrate solution (S1804, Sigma Aldrich) were added. The solution became colorless and then turned purple. After one minute, the solution was heated until it became cherry red. 9.5 ml of 1% sodium citrate (S1804, Sigma Aldrich) were added. The water was heated to boiling point and 8.6 ml of 1% gold chloride solution (G4022, Sigma Aldrich) were added. The solution became dark blue, virtually black, then purple and was then left to cool to ambient temperature.
[0198] The pH of 10 ml of the purple colloidal gold solution previously obtained was adjusted, on the one hand, using a 0.2 M potassium carbonate solution (269619, Sigma Aldrich), until a pH of 8.8 was reached; and the pH of 10 ml of purple colloidal gold solution was adjusted to pH 7.8, on the other hand, using a 0.2 M potassium carbonate solution (269619, Sigma Aldrich). 200 μg of anti-IGFBP-1, clone G2, were added to 1 ml of distilled water and then 10 ml of purple colloidal gold solution at the pH adjusted to 8.8 were rapidly added. 200 μg of anti-AIGFBP-1, clone F8, were added to 1 ml of distilled water and then 10 ml of purple colloidal gold solution at the pH adjusted to 7.8 were rapidly added. These tubes were placed on a circular mixer (multidisk rotator 5-50 rpm, Sobinco (registered trademark)) for 20 minutes. 1.5 ml of Gold Stabilization Buffer solution (Artron Bioresearch (registered trademark)) were added. The 2 tubes were centrifuged (5804, Eppendorf (registered trademark)) at 4000 rcf for 30 minutes. The supernatant was suctioned off using a vacuum pump (159600, Brand (registered trademark)) and the pellet was taken up with 1 ml of resuspension buffer (pH 8.0) containing 20 mM Tris Base (26-128-3094, Euromedex (registered trademark)), 50 mM NaCl (S7653, Sigma Aldrich (registered trademark)), 0.2% BSA (1035-70, ID Bio (registered trademark)), 10% sucrose (S8501, Sigma Aldrich), 5% trehalose (T9531, Sigma Aldrich (registered trademark)).
[0199] 825 μl of the saturating solution ((pH 7.4) 50 mM Na.sub.2HPO.sub.4 (S7907, Sigma Aldrich (registered trademark)), 1% of bovine serum albumin (BSA) (1035-70, ID Bio), 20% of sucrose (S8501, Sigma Aldrich) and 5% of trehalose (T9531, Sigma Aldrich (registered trademark))) were added to 125 μl of solution of IGFBP-1 conjugate, clone F8, to which 50 μl of IGFBP-1 conjugate, clone G2, had been added. The mixture thus obtained was stirred for 20 minutes at ambient temperature on the circular mixer adapted to 1.5 ml tubes.
[0200] The mixture of antibodies coupled to the colloidal gold particles was then sprayed, with a flow rate of 4 μl/cm, onto glass fiber sheets (sample conjugate pad zone) having dimensions of 30 cm 3.2 cm (#8964 Ahlstrom, MAPDS-0300, Arista Biologicals (registered trademark)) pretreated with a solution of 0.1 M B.sub.4Na.sub.2O.sub.7 (B3545, Sigma Aldrich (registered trademark)), 1% triton X-100 (X-100, Sigma Aldrich (registered trademark)), pH 8.4.
[0201] The binding of this conjugate mixture to the glass fiber sheet was carried out with the same Isoflow Dispenser (registered trademark) apparatus while complying with the manufacturer's indications. After spraying, the glass fiber sheets were dried for 30 minutes in an oven at 37° C. under an atmosphere at a controlled humidity of less than 30% in order to dry them.
[0202] The cards were assembled by adhesive bonding of the conjugate-impregnated glass fiber onto the lower adhesive part of the card while making the glass fiber sheet overlap by 1 to 3 mm on the nitrocellulose membrane. In the same way, a paper with a high absorption capacity (absorbent pad zone) (Ahlstrom 222, MAPDS-0100, Arista Biologicals) having dimensions of 30 cm×1.6 cm was placed on the upper part of the card and overlapped the nitrocellulose membrane by 1 to 3 mm in order to be able to create a migration flow.
[0203] The card thus assembled was cut into strips 4 mm wide using a guillotine (model CM4000, Biodot).
[0204] The strips were then placed in a plastic support called a cassette (MK001, D2 Technologies (registered trademark)).
[0205] The cassettes were then closed using a clipping device (Closure-I, A-Point Technologies (registered trademark)).
[0206]
Example 2: Example of Use of a Device for Implementing the Method of Some Embodiments
[0207] 1. A solution of 1 ml of 50 mM Na.sub.2HPO.sub.4 (S7907, Sigma Aldrich), 1% of bovine serum albumin (BSA) (1035-70, ID Bio), 0.05% triton X-100 (X-100, Sigma Aldrich (registered trademark)). 0.55% of tween 20 (P7949, Sigma Aldrich (registered trademark)), 0.1% of NaN.sub.3 (58032, Sigma Aldrich (registered trademark)) (pH-7.4) was introduced into a dropper bottle (015610, Alltest), [0208] 2. the sample was taken with a sterile swab, the bud of which is made of polyester, then deposited in the dropper bottle containing the solution. [0209] 3. the mixture obtained was introduced into the wells of the cassettes described in example 1, [0210] 4. the result is obtained after 10 minutes of migration.
[0211] The test is validated when a colored band appears at the level of the control line.
[0212] The test is positive when a colored band appears at the level of at least two test zones (IL-6, IGFBP-1 A and/or IGFBP-1B).
Example 3: Method for Detecting Imminent Birth
[0213] In this example, pregnant women who receive care in the five tertiary hospitals in the south of Nigeria were included. The study was approved by the Conseil Institutionnel Examen [Examining Institutional Council] of NAUTH (letter of approval NAUTH/cs/66/VOL 8/56), and other participating hospitals. Any person who accepted to participate in the study gave an informed consent.
[0214] The diagnosis of risk of imminent birth and of preterm birth was carried out in compliance with the protocol of the institutions, including women with intact fetal membranes, with contractions with or without increase in intensity and/or in frequency, a partially or totally effaced cervix, a cervix at less than 3 cm. Pregnant women with a gestational age of 24+0 to 36÷6 presenting symptoms or signs of risk of preterm labor were included. These women presented self declared symptoms or signs, or complaints suggestive of preterm labor.
[0215] For admission, the gestational age established on the basis of the ultrasound of the first or second trimester had to be in agreement with the menstrual dates. Women for whom there was a divergence was in particular a final menstrual period and the gestational age at the ultrasound .Math.10 days to .Math.20 weeks. The exclusion criteria were composed of multiple pregnancy, polyhydramnios, premature rupture of fetal membranes, triplets, prior cervical examination, sexual intercourse within 24 hours, tocolysis, vaginal bleeding, cervical cerclage, placenta previa, or a known congenital abnormality of the fetus, women suffering from chronic diseases (hypertension, diabetes, renal or cardiac diseases) or genital tract abnormalities were excluded. Pregnancies with delivery less than 14 days post-recruitment for maternal and/or fetal indications were also excluded.
[0216] Following the inclusion study, the women underwent a speculum examination carried out while vaginal smear samples were taken in order to measure/detect total IGFBP-1, fetal fibronectin and interleukin 6, prior to the cervical examination. The cervico-vaginal samples were collected during a sterile speculum examination using a Dacron swab by placing the lower end of the swab shaft in the posterior cul de sac of the vagina for 30 s (so as to allow saturation of the pad). After the collection, the pad was inserted into a tube containing 1 ml of extraction buffer (see
[0217] The antibodies used were: for IGFBP-1 A, anti-IGFBP-1 antibodies sold by the company Hytest (catalog number 4IG8; clone G5F8 and clone C7B9), for IGFBP-1 B, antibodies sold by the company Hytest (catalog number 4I52; clone G2; catalog number 4IG8; clone G5F8 and clone C7B9), for IL-6, the antibodies sold by Diaclone; catalog number 855.050.005, clone B-E8 or B-E4. The antibodies used for detecting the fibronectin (A and B) were polyclonal antibodies produced in rabbit, sold by the company Sigma under the reference F3648 (Sigma).
[0218] For the fibronectin and the IGFBP-1, two different forms of these proteins were used as markers: fibronectin A and fibronectin B, and IGFBP-1 A and IGFBP-1 B. After measurement, a monitoring of birth or of the possibility of birth was performed 2, 7 and 14 days later. A comparison of the results via the detection of the markers: IGFBP-1 A, IGFBP-1 B and IL-6 with the detection of fibronectin A and B was carried out. The test was considered to be positive if at least two of the three biomarkers IGFBP-1, IGFBP-1 B and IL-6 were present in the sample.
[0219] The cervical measurements were carried out by transvaginal ultrasound. During a gynecological examination, the speculum examination was carried out by introducing a Cusco speculum into the vagina before touching the vagina. The parameters recorded during the pelvic examination included cervical dilation, membrane state, cervical effacement, patient history, namely age, number of births and miscarriages, and the results of the triplex tests.
[0220] The cervical examinations preceded each ultrasound examination. To minimize the intra-observer variability of the cervical measurement, the women had to empty their bladder just before the ultrasound and the sagittal cervical canal was perfectly identified so that the internal orifice and the external orifice were both identified and the actual length corresponding to the distance corresponding to the straight line between them was measured.
Statistical Analyses
[0221] The size of the sample was calculated as being 94 with a power of 80%, .Math.=0.05, .Math.=20%, and a standard value of the effect of 0.84. The Student's t test was used to analyze the continuous variables. The statistical significance of the differences between the groups for the continuous variables was evaluated and the differences in proportions were evaluated using the Mantel-Haenszel test. The data were analyzed with Epi Info Version 3.5.1 (Centers for Disease Control and Prevention, Atlanta, Ga., USA) and Stata version 10 (StataCorp, College Station, Tex., USA). The values of P<0.05 were considered to be statistically significant.
[0222] The sensitivity and specificity, the positive and negative predictive values and the accuracy values were also determined. The sensitivity was calculated as follows: true positive (TP)/[TP+false negatives (FN)]; and the specificity was calculated as follows: true negative (TN)/[TN+false positives (FP)]. The positive predictive value (PPV)=TP/(TP+FP), while the negative predictive value NPV=TN/(TN+FN). In addition, the accuracy=([true positive+true negative]/[true negative+false negative+false positive+true negative]+100%) of each test was also estimated. The probability of a preterm birth was measured by calculating the relative risk (RR) ratio and the respective confidence intervals (CI) at 95% for each test.
[0223] 117 women with single pregnancies were evaluated for admissibility to participate in the study and 98 of them were recruited. Of the 98 women who were recruited, three women were excluded (two women had non-valid results and one woman underwent a planned Cesarean section owing to an iatrogenic factor before the 14 days of the monitoring period and their data were therefore excluded from the statistical analysis). A total of 95 women were finally included in the analysis. The model for inclusion of women participating in the study is shown in
[0224] For the women included, the average age was 31.1±2.3 years old (extremes: 20-40). The mean gravidity and the mean parity were respectively 2.5±0.9 (range of 1-6) and 1.4±0.4 (range of 0-5). The mean gestational age at inclusion was 34.1±1.8 weeks (range of 24-36 weeks).
[0225] The matrices of performance of the tests (IGFBP-1 A, IGFBP-1 B and IL-6 with respect to fibronectin) in the 2 days, 7 days and 14 days of inclusion in the study are presented respectively in table 1, table 2 and table 3. Overall, the predictive accuracy of the combined tests with respect to the days of inclusion was: 85.3% (.Math.2 days), 93.7% (.Math.7 days) and 94.7% (.Math.14 days) which is higher than the accuracy with fibronectin A. (71.6%. 82.1% and 85.3%) 75 and of fibronectin B (57.9%, 70.5% and 71.6%) respectively.
[0226] Of the 95 patients included in the analysis, 25 (26.3%) gave birth before 37 weeks of gestation and 70 (73.7%) gave birth at 37 weeks of gestation or later. The matrix of performance of IGFBP-1 A/IGFBP-1 B and IL-6 with respect to fibronectin as a function of the delivery time is described in table 4.
[0227] The mean body temperature for the women included in the study was 36.8±0.4° C. The mean cervical length by ultrasound for the women who gave birth in the 14 days following inclusion in the study was 24.4±1.3 mm compared with 32.5±1.9 mm in the women who did not give birth in the 14 days following inscription (T=−34.29; p<0.001).
TABLE-US-00004 TABLE 1 Results of the combination IGFBP-1 A/IGFBP-1 B and IL-6 with respect to fibronectin (A/B) of the tests in women in preterm labor with a risk of giving birth at * 2 days after inclusion in the study 2/3 among Fibonectin Fibonectin IGFRP-1 A B A/ (detection (detection IGFBP-1 threshold threshold IGEBP- IGFBP- Markers B & IL-6 500 ng/ml) 50 ng/ml) I A 1 B IL-6 Criteria True 62 51 38 68 61 52 negative True 19 17 17 16 18 19 positive False 0 3 2 4 2 1 negative 14 25 38 9 14 24 False positive Total 95 95 95 95 95 95 % 81.6 67.1 50 88.3 81.3 68.4 speci- ficity Sensi- 100 85 89.5 80 90 95 tivity Negative 100 94.4 95 94.4 96.8 98.1 predictive value Positive 57.6 40.5 30.9 64 56.3 44.2 predictive value Accuracy 85.3 71.6 57.9 88.4 83.2 74.7
[0228] The accuracy of 85.3% was statistically significant with respect to the accuracy of fibronectin A of 71.6% (Mantel-Haenszel chi squared=5.23, p=0.02, relative risk=1.59, CI at 95%=01.02 to 02.25) or of fibronectin B of 57.9% (Mantel-Haenszel chi squared=17.40, p<0.001, risk ratio=2.30, CI at 95%=1.43 to 3.68).
TABLE-US-00005 TABLE 2 Results of the combination IGFBP-1 A/IGFBP-1 B and IL-6 with respect to fibronectin (A/B) of the tests in the women in preterm labor with a risk of giving birth at * 7 days after inclusion in the study 2/3 among Fibronectin Fibronectin IGFBP-1 A B A/ (detection (detection IGFBP-1 threshold threshold IGFBP- IGFBP- Markers B & IL-6 500 ng/ml) 50 ng/ml) 1A 1B IL-6 Criteria True 60 50 38 63 60 50 negative True 29 28 29 23 28 29 positive False 4 3 2 8 2 2 negative False 4 14 26 1 5 14 positive Total 95 95 95 95 95 95 % 93.8 78.1 59.4 98.4 96.8 78.1 speci- ficity Sensi- 87.9 90.3 93.5 74.2 93.3 96.2 tivity Negative 93.8 94.3 95 88.7 96.8 96.2 predictive value Positive 87.9 66.7 52.7 95.8 84.8 67.4 predictive value Accuracy 93.7 82.1 70.5 90.5 92.6 83.2
[0229] The accuracy of 93.7% was statistically significant with respect to the accuracy of fibronectin A of 82.1% (Mantel-Haenszel chi squared=5.95, p=0.01, relative risk=2.04, CI at 95%=1.01 to 4.12) or of fibronectin B of 70.5% (Mantel-Haenszel chi squared=17.25, p<0.001, the risk ratio=3.23, CI at 95%=1.54 to 6.77).
TABLE-US-00006 TABLE 3 Results of the combination IGFBP-1 A/IGFBP-1 B and IL-6 with respect to fibronectin (A/B) of the tests in the women in preterm labor with a risk of giving birth at * 14 days after inclusion in the study 2/3 among Fibronectin Fibronectin IGFBP-1 A B A/ (detection (detection IGFBP-1 threshold threshold IGFBP- IGFBP- Markers B & IL-6 500 ng/ml) 50 ng/ml) 1 A 1 B IL-6 Criteria True 59 50 37 61 59 49 negative True 31 31 31 23 31 31 positive False 3 3 3 10 3 3 negative False 2 11 21 1 2 12 positive Total 95 95 95 95 95 95 % 96.7 82 60.7 98.4 96.7 80.3 speci- ficity Sensi- 94.2 91.2 91.2 69.7 91.2 94.2 tivity Negative 93.9 73.8 56.4 95.8 93.9 72.1 predictive value Positive 93.9 73.8 56.4 95.8 93.9 72.1 predictive value Accuracy 94.7 85.3 71.6 88.4 94.7 84.2
[0230] The accuracy of 94.7% was statistically significant with respect to the accuracy of fibronectin A of 85.3% (Mantel-Haenszel chi squared=4.71, p=0.03, relative risk=2.00, CI at 95%=0.93 to 4.30) or of test fibronectin B of 71.6% (Mantel-Haenszel chi squared=18.09, p<0.001, risk ratio=3.65, CI at 95%=1.61 to 8.25).
TABLE-US-00007 TABLE 4 Result of the combination with respect to the fibronectin test on the cases of preterm labor as a function of gestational age for the women having given birth at * 14 days after inclusion in the study 2/3 among Fibronectin Fibronectin IGFBP-1 A B A/ (detection (detection IGFBP-1 threshold threshold IGFBP- IGFBP- Markers B & IL-6 500 ng/ml) 50 ng/ml) 1 A 1 B 1L-6 Criteria True 59 50 37 61 59 49 negative True 31 31 31 23 31 31 positive False 3 3 3 10 3 3 negative False 2 11 21 1 2 12 positive Total 95 95 95 95 95 95 % 96.7 82 60.7 98.4 96.7 80.3 speci- ficity Sensi- 91.2 91.2 91.2 69.7 91.2 91.2 tivity Negative 95.2 94.3 92.5 85.9 95.2 94.2 predictive value Positive 93.9 73.8 56.4 95.8 93.9 72.1 predictive value Accuracy 94.7 85.3 71.6 88.4 94.7 84.2
[0231] Of the 95 patients included in the analysis. 25 (26.3%) gave birth before 37 weeks of gestation and 70 (73.7%) gave birth at or after 37 weeks of gestation. The matrix of performance of IGFBP-1 A/IGFBP-1B and IL-6 with respect to fibronectin as a function of the delivery date is shown in table 2.
[0232] The mean body temperature for the women was 36.8±0.4° C. The mean cervical length by ultrasound for the women who gave birth in the 14 days following inclusion was 24.4±1.3 mm compared with 32.5±1.9 mm in the women who did not give birth in the 14 days following inscription (T=−34.29; p<0.001).
[0233] As demonstrated in this example, the method of some embodiments in which at least two of the markers are positive makes it possible to obtain results which are more sensitive and more specific with a better positive and negative predictive value than the known reference test using fibronectin [13]. In particular, the method according to some embodiments makes it possible to obtain results with a positive predictive value which is much higher than that using fibronectin.
[0234] In particular, as demonstrated in this example, the method of some embodiments makes it possible to obtain a strong sensitivity of 91.2%, with an accuracy and the specificity, positive predictive value and negative predictive value of the combined, test which are higher, for example 25% higher, than for fibronectin A or fibronectin B respectively. The differences in results were statistically significant (p<0.05).
[0235] In addition, as demonstrated in this example, the method makes it possible to obtain a sensitivity equal to 100% and a negative predictive value of 100% for a threat of giving birth at two days.
[0236] As demonstrated in this example, the method according to some embodiments makes it possible, by virtue of its sensitivity, specificity, and negative and positive predictive value, to accurately identify the women who will experience spontaneous preterm labor.
Specifically, the sensitivity is 91.2% and the specificity is 96.7%. The high negative predictive value, 93.9%, combined with the strong sensitivity and specificity, makes it possible to avoid needless medical obstetric procedures during the pregnancy.
[0237] In addition, in the case of a full-term pregnancy, the sensitivity and the specificity were respectively 81.8% and 96.6%, clearly demonstrating that the method according to some embodiments is a method that is of use for predicting a full-term pregnancy.
[0238] In addition, an analysis of the results when the three markers are positive and/or negative as a function of the date of the birth was carried out and is presented in table 5 below.
TABLE-US-00008 TABLE 5 Result of the test for the women who had or had not given birth at less than 14, 7 and 2 days after inclusion in the study Birth < 14 days No Yes Total IGFBP-1 A + Triple negative 48 1 49 IGFBP-1 B + Triple positive 1 23 24 IL-6 Other result 17 10 22 Total 61 34 95 Birth < 7 days No Yes Total IGFBP-1 A + Triple negative 48 1 49 IGFBP-1 B + Triple positive 1 23 24 IL-6 Other result 16 6 22 Total 65 30 95 Birth < 2 days No Yes Total IGFBP-1 A + Triple negative 49 0 49 IGFBP-1 B + Triple positive 14 10 24 IL-6 Other result 19 3 22 Total 82 13 95 Results with a triple positive Birth or triple negative result < 14 days < 7 days < 2 days IGFBP-1 A + Sensitivity 95.8% 95.8% 100.0% IGFBP-1 B + Specificity 98.0% 98.0% 77.8% IL-6 NPV 98.0% 98.0% 100.0% PPV 95.8% 95.8% 41.7% Other result 23.2% 23.2% 23.2%
[0239] As demonstrated below, the method of some embodiments advantageously makes it possible to obtain a result with a sensitivity and a specificity equal respectively to 95.8% and 98.0% for a threat of giving birth at 7 or 14 days.
[0240] In addition, when the result is triple negative, the negative predictive value for a birth at 7 or 14 days is equal to 98%, and 100% for a birth at 2 days.
[0241] Thus, this example clearly demonstrates that the determination of the presence or absence in the vaginal secretions of at least two markers chosen from IGFBP-1 (A and B) and IL-6 is a reliable method for predicting imminent birth in women in preterm labor and has a sensitivity and a specificity for the prediction of imminent birth much higher than the reference methods used clinically. In addition, this example clearly demonstrates that the use of IGFBP-1 and of IL-6 makes it possible to maximize the sensitivity and to minimize the false-positive diagnoses. Furthermore, the method according to some embodiments makes it possible to obtain a “tare” negative with a negative predictive value equal to 98% or 100%, thus making it possible to avoid needless clinical procedures and or pharmacological treatments.
Example 4: Method for Detecting Imminent Birth
[0242] In this example, the protocols for admission, for carrying out the test and for examinations after inclusion and the women included were identical to those of example 3 above.
[0243] The search for IGFBP-1 A, IGFBP-1 B and IL-6 was carried out as described in example 3 above.
[0244] When IGFBP-1 A was detected, a value I.sub.A equal to 2 was assigned; when IGFBP-1 B was detected, a value I.sub.B equal to 2 was assigned and when IL-6 was detected, a value I.sub.6 equal to 1 was assigned. In the absence of detection, the value assigned was 0 for each of the markers.
[0245] After assignment of the I.sub.A, I.sub.B and I.sub.6 values, the values were added together according to the score S.sub.1 with the following formula:
S.sub.1=I.sub.A+I.sub.B+I.sub.6
[0246] The values of the score S.sub.1 obtained for the women who had or had not given birth at less than 14, 7 and 2 days after inclusion in the study are summarized in table 6 below.
TABLE-US-00009 TABLE 6 Result of the test for the women who had or had not given birth at less than 14, 7 and 2 days after inclusion in the study Number of patients having given birth Total Number of Under Under Under score S.sub.1 patients 2 days 3-7 days 8-14 days 0 50 0 1 0 1 12 1 0 0 1-4 11 2 4 4 5 24 16 7 0
[0247] On the basis of the results described in table 6 above, the calculated probabilities of giving birth were calculated from the results obtained as a function of the score obtained and are summarized in table 7 below.
TABLE-US-00010 TABLE 7 Probability of giving birth as a function of the score Number Probability of giving birth Score of Under 2 Under 7 Under 14 S.sub.1 patients days days days 0 50 0% 2% (1/50) 2% (1/50) 1 12 8.30% (1/2) 8.30% (1/12) 8.30% (1/12) 1-4 11 18.20% (2/11) 54.50% (6/11) 90.90% (10/11) 5 24 66.70% (16/24) 95.80% (23/24) 95.80% (23/24)
[0248] As illustrated in table 7 above, a score S1 greater than or equal to 2 implies a probability of giving birth within the next 14 days of greater than 90.90%.
[0249] The negative and positive predictive values were calculated according to the following formulae:
PPV=TP/(TP+FP) and NPV=TN/(TN+FN)
in which TP signifies true positive, FP signifies false positive. TN signifies true negative and FN signifies false negative.
[0250] In addition, an evaluation of the negative and positive predictive value when the score S1 obtained was greater than or equal to 2 as a function of the time until birth, less than or equal to 2 days, less then or equal to 7 days or less than or equal to 14 days, was carried out. Table 8 below summarizes the results obtained.
TABLE-US-00011 TABLE 8 Negative or positive predictive values with a score greater than or equal to 2 Time until birth Less than Less than Less than or equal to or equal to or equal to Score S.sub.1 .Math. 2 2 days 7 days 14 days Positive 51.4% 82.9% 94.3% predictive value (PPV) Negative 98.4% 96.8% 96.8% predictive value (NPV)
[0251] As demonstrated in table 8 above, regardless of the imminent nature of the birth, for example less than 2 days, less than 7 days and/or less than 14 days, when the score S.sub.1 is greater than or equal to 2, the negative predictive value is greater than 96.8% ranging to 98.4%.
[0252] An evaluation of the negative predictive value when the score S1 obtained was equal to 0 or of the positive predictive value when the score S1 obtained was equal to 5 as a function of the time until birth, less than or equal to 2 days, less than or equal to 7 days or less than or equal to 14 days, was carried out. Table 9 below summarizes the results obtained.
TABLE-US-00012 TABLE 9 Negative predictive value with a score equal to 0 and positive predictive value with a score equal to 5 Time until birth Score S.sub.1 .Math. 2 days .Math. 7 days .Math. 14 days 5 PPV 66.7% 95.8% 95.8% 0 NPV 100.0% 98.0% 98.0%
[0253] In addition, as demonstrated in table 9 above, the method makes it possible to obtain, a negative predictive value of 100% for a threat of giving birth at two days and equal to 98% for giving birth in the next 14 days.
[0254] The example is also carried out with a different assignment of values for each of the markers IGFBP-1 A, IGFBP-1 B and IL-6. Table 10 below summarizes the various assignments, distinguishing values of the score and associated results.
TABLE-US-00013 TABLE 10 Assignment of the values and calculation of the corresponding scores Time until birth Less than or Less than or Less than or Markers Present Absent S.sub.1 = I.sub.A + I.sub.B + I.sub.6 equal to 2 days equal to 7 days equal to 14 days IGFBP-1 A 2 0 S1 = 0 NPV 100.0% 98.0% 98.0% I.sub.A IGFBP-1 B 2 0 S.sub.1 .Math. 2 PPV 51.4% 82.9% 94.3% I.sub.B S.sub.1 < 2 NPV 98.4% 96.8% 96.8% IL-6 1 0 S1 = 5 PPV 66.7% 95.8% 95.8% I.sub.6 Markers Present Absent S.sub.1 = I.sub.A + I.sub.B + I.sub.6 2 days 7 days 14 days IGFBP-1 A 3 0 S1 = 0 NPV 100.0% 98.0% 98.0% I.sub.A IGFBP-1 B 2 0 S.sub.1 .Math. 2 PPV 51.4% 82.9% 94.3% I.sub.B S.sub.1 < 2 NPV 98.4% 96.8% 96.8% IL-6 1 0 S1 = 6 PPV 66.7% 95.8% 95.8% I.sub.6 Markers Present Absent S.sub.1 = I.sub.A + I.sub.B + I.sub.6 2 days 7 days 14 days IGFBP-1 A 3 0 S1 = 0 NPV 100.0% 98.0% 98.0% I.sub.A IGFBP-1 B 3 0 S.sub.1 .Math. 3 PPV 51.4% 82.9% 94.3% I.sub.B S.sub.1 < 3 NPV 98.4% 96.8% 96.8% IL-6 1 0 S1 = 7 PPV 66.7% 95.8% 95.8% I.sub.6 Markers Present Absent S.sub.1 = I.sub.A + I.sub.B + I.sub.6 2 days 7 days 14 days IGFBP-1 A 4 0 S1 = 0 NPV 100.0% 98.0% 98.0% I.sub.A IGFBP-1 B 3 0 S.sub.1 .Math. 3 PPV 51.4% 82.9% 94.3% I.sub.B S.sub.1 < 3 NPV 98.4% 96.8% 96.8% IL-6 1 0 S1 = 8 PPV 66.7% 95.8% 95.8% I.sub.6
[0255] As demonstrated in his example, the method according to some embodiments makes it possible, by virtue of its sensitivity, specificity, negative predictive value and positive predictive value, to accurately identify the women who will give birth within a predefined period of time.
[0256] In addition, from the very high negative predictive value, namely greater than 98% or even equal to 100%, the method according to some embodiments makes it possible to reduce the false-negative results and therefore provides a reliable result which can be used by clinicians.
[0257] As demonstrated in this example, the method according to some embodiments makes it possible, by virtue of its sensitivity, specificity, negative predictive value and positive predictive value, to accurately identify the women who will give birth within a predefined period of time, making it possible to avoid needless hospitalizations and medical obstetric procedures during the pregnancy.
[0258] Thus, this example clearly demonstrates that the method including the calculation of the score S.sub.3 according to some embodiments is a reliable method for predicting imminent birth in women in preterm labor and has a sensitivity and a specificity for predicting imminent birth which are much higher than the reference methods used clinically.
[0259] Furthermore, this example clearly demonstrates that a method according to some embodiments makes it possible to obtain a “true” negative with a negative predictive value equal to 98% or 100%, thus making it possible to avoid needless hospitalizations, clinical procedures and/or pharmacological treatments.
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