Device for noninvasive continuous monitoring of premature rupture of fetal membranes and preparation method thereof

Abstract

The invention discloses a device for noninvasive continuous monitoring of premature rupture of fetal membranes and a preparation method, and relates to the field of pregnancy and delivery detection.The device comprises a reagent strip and a protection pad, the protection pad is formed by compounding a surface, a water absorption layer and an isolation layer from top to bottom, the reagent strip is arranged between the surface and the water absorption layer in the long edge direction of the protection pad, and the reagent strip is arranged between the surface and the water absorption layer in the chromatography direction. Four functional areas, namely a sampling area, a reaction area, an observation area and a recovery area, are adhered in sequence; the sampling area is a sample pad; the reaction area is a combination pad and is dried with an anti-human IGFBP-1 monoclonal antibody 2-latex microsphere compound; the observation area is provided with a nitrocellulose membrane, a detection line is fixed on the nitrocellulose membrane, and the detection line is composed of an anti-human IGFBP-1 monoclonal antibody 1, a buffer system and a quality control dye; the recovery area is made of absorbent paper. Whether amniotic fluid leakage occurs or not can be accurately judged, meanwhile, the protection pad uses a special quality control system to replace a quality control line of a traditional reagent strip, and the result is clearer and easier to judge.

Description

Technical Field

[0001] The invention belongs to the field of pregnancy and childbirth detection, and specifically relates to a device for non-invasive and continuous monitoring of premature rupture of fetal membranes and a preparation method thereof. Background Art

[0002] The existing detection of premature rupture of membranes is mainly through pH test paper, vaginal fluid smear microscopy, ultrasound-assisted diagnosis, etc. pH test paper has a high false positive rate and is easily interfered by human body fluids; vaginal fluid smear microscopy is complicated to operate, and the interpretation of the results is affected by the experience of the tester.

[0003] In order to improve accuracy, it is becoming a trend to predict whether pregnant women will have premature rupture of membranes by testing special biological indicators: such as cervical and vaginal secretions to detect alpha-fetoprotein (AFP), human placental lactogen (HPL), cervical secretions to detect fetal fibronectin (FFN), cervical secretions to detect insulin-like growth factor binding protein (IGFBP-1), placental alpha microglobulin (PAMG-1) and soluble intercellular adhesion molecule (sICAM-1) in cervical secretions. However, biological indicator testing usually requires swabs to be inserted deep into the vagina to collect fluid for testing, which requires professional operation. At the same time, intermittent leakage of amniotic fluid or high-level rupture of water may make it impossible to accurately collect samples. Therefore, considering the user experience and biosafety, it is not conducive to the promotion of self-testing at home. Summary of the invention

[0004] The purpose of the present invention is to provide a non-invasive rapid premature rupture of membranes detection device and preparation method. A sanitary pad containing a reagent strip, the reagent strip is prepared based on immunochromatography, the pad continuously collects vaginal discharge from pregnant women for detection, and finally reflects the result through a visual signal.

[0005] Provided is a pad for premature rupture of membranes detection, which can quickly distinguish between amniotic fluid and urine and accurately determine whether amniotic fluid leakage occurs, while facilitating timely detection and sampling.

[0006] The technical solution adopted by the present invention is:

[0007] The first purpose of the embodiment of the present invention is to provide a device for non-invasive continuous monitoring of premature rupture of membranes, including a reagent strip and a pad, wherein the pad is composed of a surface layer, a water-absorbing layer, and an isolation layer from top to bottom, and the reagent strip is placed between the surface layer and the water-absorbing layer along the length direction of the pad, and the reagent strip is sequentially adhered to four functional areas along the chromatography direction: a sampling area, a reaction area, an observation area, and a recovery area; the sampling area is a sample pad; the reaction area is a binding pad: an anti-human IGFBP-1 monoclonal antibody 2-latex microsphere complex is dried, and the purpose is to detect IGFBP-1 in the sample. When the sample passes through the sampling area, the IGFBP-1 contained therein will form an IGFBP-1-anti-human IGFBP-1 monoclonal antibody 2-latex microsphere complex with the anti-human IGFBP-1 monoclonal antibody 2-latex microsphere, which can then be captured and colored by the detection line.

[0008] The observation area is provided with a nitrocellulose membrane, and the nitrocellulose membrane is fixed with a detection line, which is composed of anti-human IGFBP-1 monoclonal antibody 1, a buffer system and a quality control dye, and is used to capture the IGFBP-1-anti-human IGFBP-1 monoclonal antibody 2-latex microsphere complex, which is the site of immune reaction.

[0009] The recovery area is absorbent paper.

[0010] As a preferred solution of the embodiment of the present invention, the bottom of the above-mentioned reagent strip is provided with a self-adhesive bottom plate, the outer periphery of the reagent strip is wrapped with waterproof material, and the size of the reagent strip is 40*5-50*10mm; preferably, the size of the reagent strip is 45*8mm.

[0011] As a preferred solution of the embodiment of the present invention, the waterproof material is a transparent polyolefin film.

[0012] As a preferred solution of the embodiment of the present invention, the sample pad is soaked and dried with a diluent, and the diluent includes a buffer system, a blocking protein, and a surfactant, and is used to absorb the sample and eliminate matrix interference.

[0013] As a preferred solution of the embodiment of the present invention, the absorbent paper material includes pulp and fluff pulp, which mainly provides chromatography power and recovers waste liquid.

[0014] The second purpose of the embodiment of the present invention is to provide a method for preparing a device for non-invasive continuous monitoring of premature rupture of membranes, comprising the following steps:

[0015] 1) Cutting and pasting of absorbent paper, nitrocellulose membrane, conjugate pad and sample pad:

[0016] 2) Preparation, coating and drying of test lines;

[0017] 3) Preparation, coating and drying of antibody-latex microsphere complexes;

[0018] 4) Sample pad solution preparation, soaking and drying;

[0019] 5) Bonding of absorbent paper, nitrocellulose membrane, conjugate pad and sample pad;

[0020] 6) Cutting, laminating, assembling and packaging.

[0021] As a preferred solution of an embodiment of the present invention, the absorbent paper is cut into 1-1.2*30cm / strip, the nitrocellulose membrane is cut into 1-1.2*30cm / strip, the conjugate pad is cut into 0.8-1*30cm / strip, and the sample pad is cut into 2.5*30cm / strip.

[0022] As a preferred embodiment of the present invention, in the above step 2), PBS is used to dilute the anti-human IGFBP-1 monoclonal antibody to 0.5 to 2 mg / mL, and a quality control dye is added to a final concentration of 0.025 to 0.05 mg / mL, and coated on a nitrocellulose membrane at 1.0 to 2.0 μL / cm, and dried at 50°C overnight for use.

[0023] As a preferred embodiment of the present invention, the quality control dye is any one or any combination of rhodamine B, methyl red, phenol red, cresol red, and neutral red.

[0024] As a preferred solution of an embodiment of the present invention, the above step 3) further comprises the activating agent being 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride.

[0025] As a preferred solution of an embodiment of the present invention, the above-mentioned covering film covers the peripheral area of ​​the sampling area, and the peripheral edge of the sample pad is overlapped with the water absorption layer as a whole.

[0026] As a preferred solution of an embodiment of the present invention, the sampling area is a circle with a diameter ≥ 18 mm or a square with a side length ≥ 18 mm.

[0027] As a preferred solution of the embodiment of the present invention, the sampling area is a square with a size of 18mm*18mm, or a circle with a diameter of 18mm. ;

[0028] As a preferred solution of the embodiment of the present invention, the size of the above-mentioned bonding pad is a quadrilateral with a size of 8mm*5mm-8mm*6mm. Compared with the prior art, the advantages of the present invention are:

[0029] 1) The protective pad for accurate detection of premature rupture of membranes in the present invention uses IGFBP-1 as a marker to detect whether vaginal fluid contains amniotic fluid to determine the condition of premature rupture of membranes. It has high specificity and can effectively eliminate the interference of urine and other liquids in the detection, thereby improving the accuracy of amniotic fluid detection and reducing the probability of false positive detection.

[0030] 2) The discomfort caused by existing reagent tests is reduced. The reagent strip is placed on the water-absorbing layer, and the materials of each component are optimized based on the basic principle of softness and thinness, and the key parts of the reagent strip are waterproofed; in order to reduce the foreign body sensation and make it suitable for long-term wear, the shape of the reagent strip is optimized from slender to short and wide, and the size is optimized from 60*4mm to 45*8mm, reducing edge discomfort while also widening the observation window to make the results easier to observe;

[0031] 3) The pad uses a special quality control system to replace the quality control line used in traditional reagent strips, making the results clearer and easier to judge. Since the overall length of the reagent strip is shortened, we merge the coated quality control system with the detection system. Different from the traditional detection method that judges the validity of the result by the presence or absence of the quality control line, the reagent strip in the present invention prompts the result by the color change of the line in the observation area.

[0032] 4) In order to minimize the sample volume required for testing, the hollow area of ​​the sampling area film is increased. Due to the waterproofness of the film material, after the sample contacts the surface material, it mainly diffuses rather than infiltrates. By increasing the hollow area of ​​the sampling area film, the sampling area of ​​the reagent strip is fully in contact with the water-absorbing layer of the pad, and the water-absorbing layer of the pad increases the water-absorbing area and power to increase the force of sample infiltration. By balancing the sample diffusion and infiltration forces, the area of ​​the sampling area of ​​the reagent strip should be designed to be ≥18mm in diameter or side length, so that the volume of the sample that completes the chromatography is reduced to 120μL, effectively reducing the interference of the pad system. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic structural diagram of a pad for accurate detection of premature rupture of membranes in an embodiment of the present invention;

[0034] Figure 2 for Figure 1 A top view of an embodiment;

[0035] Figure 3 It is a schematic diagram of the structure of the test strip;

[0036] Figure 4 This is a schematic diagram of a negative test result;

[0037] Figure 5 This is a schematic diagram of a positive test result;

[0038] Figure 6 It is a schematic diagram of invalid detection results;

[0039] Figure 7 Schematic diagram of packaging method 1 of the reagent strip;

[0040] Figure 8Schematic diagram of packaging method 2 of the reagent strip;

[0041] Fig. 9 Schematic diagram of packaging method 3 of the reagent strip;

[0042] Fig.10 Schematic diagram of packaging method 4 of the reagent strip.

[0043] In the figure: 10 - reagent strip, 11 - sampling area, 12 - reaction area, 13 - observation area, 131 - detection line, 14 - recovery area, 20 - pad, 21 - surface layer, 22 - water absorption layer, 23 - isolation layer. Specific implementation plan

[0044] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] IGFBP-1 (insulin-like growth factor binding protein-1) is a protein with a molecular weight of 25kDa. It is synthesized and secreted by fetal or human liver cells and maternal decidua in the human body. After pregnancy, the concentration of IGFBP-1 begins to increase. It is one of the most important proteins in the amniotic fluid from the second trimester to before delivery. When the fetal membrane ruptures, the IGFBP-1 in the amniotic fluid can flow into the cervix and vagina through the rupture. The concentration of IGFBP-1 in amniotic fluid is 100 to 1000 times higher than that in urine and blood. Even a small amount of side leakage can be detected. Therefore, detecting the leakage of IGFBP-1 can effectively determine whether premature rupture of membranes has occurred.

[0046] The detection principle of the present invention is as follows: when the sample is chromatographed to the binding pad in the reaction zone, IGFBP-1 in the sample will bind to the anti-human IGFBP-1 monoclonal antibody-2-latex microsphere complex on the binding pad to form an IGFBP-1-anti-human IGFBP-1 monoclonal antibody-2-latex microsphere complex, and continue to be chromatographed, and will be captured by the anti-human IGFBP-1 monoclonal antibody-1 at the detection line in the observation zone to form an anti-human IGFBP-1 monoclonal antibody-1-IGFBP-1-anti-human IGFBP-1 monoclonal antibody-2-latex microsphere complex, thereby a blue line appears on the detection line in the observation zone, and at the same time, the quality control dye continues to be chromatographed with the sample, and is finally recovered by the absorbent paper, the purple color fades, and at this time, a blue line appears in the observation zone.

[0047] If the sample does not contain IGFBP-1 or the concentration is lower than the detection limit of the reagent strip, no visible blue line will be formed in the observation area, but the quality control dye will still be chromatographed with the sample and eventually recovered by the absorbent paper, and the purple color will fade, and there will be no lines in the observation area.

[0048] If the sample volume is insufficient to complete the chromatography, no liquid will be chromatographed to the detection line in the observation area, and the quality control dye will still exist on the reaction line in the observation area. At this time, there will be a purple line in the observation area.

[0049] Embodiment 1:

[0050] like Figure 1-Figure 3 The device for non-invasive continuous monitoring of premature rupture of membranes comprises a reagent strip and a pad, wherein the pad is composed of a surface layer, a water-absorbing layer and an isolation layer from top to bottom, the reagent strip is placed between the surface layer and the water-absorbing layer along the length direction of the pad (20), the bottom of the reagent strip is provided with a self-adhesive base plate, the self-adhesive base plate is a satin matt white polyolefin film, the outer periphery of the reagent strip is wrapped with a waterproof material of a satin matt transparent polyolefin film, and the size of the reagent strip 10 is 45*8mm. The reagent strip is sequentially adhered to four functional areas along the chromatography direction: a sampling area, a reaction area, an observation area, and a recovery area; the sampling area is a sample pad, which is formed by soaking and drying in a diluent, and the diluent includes a buffer system, a blocking protein, and a surfactant; the reaction area is a binding pad: an anti-human IGFBP-1 monoclonal antibody 2-latex microsphere complex is dried, and the purpose is to detect IGFBP-1 in a sample. When the sample passes through the sampling area, the IGFBP-1 contained therein will form an IGFBP-1-anti-human IGFBP-1 monoclonal antibody 2-latex microsphere complex with the anti-human IGFBP-1 monoclonal antibody 2-latex microsphere, which can then be captured and colored by the detection line. The observation area is a nitrocellulose membrane, on which the detection line is fixed. The detection line is composed of anti-human IGFBP-1 monoclonal antibody 1, a buffer system, and a quality control dye. The recovery area is absorbent paper, which is made of pulp or fluff pulp. Its function is to provide chromatography power and recover waste liquid.

[0051] A method for preparing a device for non-invasive continuous monitoring of premature rupture of membranes comprises the following steps:

[0052] 1) Cutting and pasting of absorbent paper, nitrocellulose membrane, conjugate pad and sample pad: cut absorbent paper into 1*30cm / strip, cut nitrocellulose membrane into 1*30cm / strip, cut conjugate pad into 0.8*30cm / strip, and cut sample pad into 2.5*30cm / strip;

[0053] 2) Preparation, coating and drying of the test line: dilute the anti-human IGFBP-1 monoclonal antibody to 1 to 1 mg / mL using PBS (containing 1% sucrose), add rhodamine B to a final concentration of 0.025 mg / mL, coat on a nitrocellulose membrane at 1.0 μL / cm, and dry at 50°C overnight for use;

[0054] 3) Preparation, coating and drying of antibody-latex microsphere complex: Take 0.1 mL of latex microspheres (4%), wash twice with 1 mL of 0.1 M MES buffer solution, add activator 1-ethyl-3 [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), the amount of activator is 100 μg per 1 mg of microspheres, then shake at room temperature for 1 hour, centrifuge to remove the supernatant, wash the latex microspheres twice with 1 mL of 0.1 M MES buffer solution, add 1 mL of 0.1 M MES buffer solution to resuspend the latex microspheres, and add 1 mg of anti-human IGFBP-1 monoclonal antibody 2 at the same time, shake at room temperature for 0.5 h, and then add 0.5 mL of 10% BSA to block for 1 h. After blocking, the supernatant was removed by centrifugation, and the microspheres were diluted to a concentration of 1 mg / mL by adding 20 mM Tris, 1% BSA, 10% sucrose, and 5% trehalose (pH 9.0). The microspheres were sprayed onto the conjugate pad at a spray volume of 2.0 μL / cm and dried at 50°C overnight for use.

[0055] 4) Preparation, soaking and drying of sample pad solution: 2.42 g Tris, 5 g Tween-20, 5 g BSA, and 10 g NaCl were dissolved in 1 L purified water to prepare a soaking solution (pH adjusted to 9.0 ± 0.1), which was then soaked in a blank glass fiber and dried at 50°C overnight for later use;

[0056] 5) Bonding of absorbent paper, nitrocellulose membrane, conjugate pad and sample pad;

[0057] 6) Cutting, laminating, assembling and packaging.

[0058] Embodiment 2:

[0059] Specificity test:

[0060] 1) Urine matrix: In view of the special population of pregnant women, the main interference condition for vaginal discharge detection is urine leakage. The results of testing urine samples from 108 healthy pregnant women and 92 non-pregnant women are shown in Table 1.

[0061] Table 1. Urine sample test results

[0062]

[0063] Result analysis: The negative compliance rate of urine samples from non-pregnant women was 100% (92 / 92) and the negative compliance rate of healthy pregnant women was 98.15% (106 / 108). The total negative compliance rate was 99%, indicating that the reagent was interfered by urine samples at a low level.

[0064] 2) Vaginal secretion matrix: Vaginal discharge samples were tested. The second interfering factor was vaginal secretion. Vaginal secretion samples from 108 healthy pregnant women were taken with swabs and soaked in 1 mL PBS buffer. After sufficient extraction, the swabs were discarded and 200 μL of liquid was taken for testing. The results were observed for 15 minutes. The results are shown in Table 2.

[0065] Table 2. Vaginal secretion sample testing

[0066]

[0067] Result analysis: The negative coincidence rate of the above test samples of vaginal secretions of healthy pregnant women was 100% (108 / 108), indicating that the reagent was not interfered by vaginal secretions.

[0068] 3) Interference from blood: Given the testing site of the reagent, the reagent is susceptible to interference from blood. Blood also contains IGFBP-1 protein, but the concentration is 100 to 1000 times lower than that in amniotic fluid. Venous blood samples from 108 pregnant women were collected using a vacuum venous sampling tube. 10 μL of venous whole blood was dripped onto the sample pad of the pad, and then 190 μL of PBS buffer was dripped to simulate the testing scenario with a small amount of bleeding. The results were observed in 15 minutes. The results are shown in Table 3.

[0069] Table 3. Blood sample tests

[0070]

[0071] Result analysis: The above test of healthy pregnant women's blood samples showed a negative compliance rate of 100% (108 / 108), indicating that the reagent is not affected by a small amount of blood samples.

[0072] Embodiment 3:

[0073] Minimum detection limit detection

[0074] The concentration of the capture antibody has a significant effect on the detection limit. The concentrations of the capture antibody were set to 0.5 mg / mL, 1 mg / mL, and 2 mg / mL. IGFBP-1 recombinant protein and amniotic fluid samples were tested using PBS buffer as the matrix. After gradient dilution, 200 μL of the test liquid was added to the sampling area and the results were observed for 15 minutes. The test results are shown in Tables 4 and 5 below:

[0075] Table 4. IGFBP-1 recombinant protein detection results

[0076]

[0077] Table 5. Amniotic fluid test results

[0078] Dilution ratio 1 / 20 1 / 50 1 / 100 1 / 200 1 / 500 1 / 1000 1 / 2000 1 / 5000 0.5mg / mL Positive Positive Positive Positive Positive Negative Negative Negative 1mg / mL Positive Positive Positive Positive Positive Positive Negative Negative 2mg / mL Positive Positive Positive Positive Positive Positive Negative Negative

[0079] Result analysis: In the results of Table 5-6 above, when the capture antibody concentration was 0.5 mg / mL, the detection limit was 20 ng / mL of IGFBP-1 recombinant protein, and the dilution multiple of amniotic fluid was 500 times. When the capture antibody concentration was 1 mg / mL and 2 mg / mL, the detection limit was 10 ng / mL of IGFBP-1 recombinant protein, and the dilution multiple of amniotic fluid was 1000 times.

[0080] Embodiment 4:

[0081] Reagent strip packaging

[0082] Since the test strip is susceptible to moisture and contamination, the product of the present invention uses waterproof and breathable materials to package the test strip. Through the study of different packaging methods and sizes ( Figure 7-Figure 10 ) to select the appropriate packaging form. The packaging principle is to expose only the sampling area of ​​the reagent strip, and the rest of the functional area is covered by waterproof material:

[0083] Lamination method such as Figure 7 , 9, and 10 are packaging methods 1, 3, and 4, respectively. The sampling area is completely exposed, and the edge of the sampling area is in contact with the water-absorbing layer. Only the overall appearance is different. Packaging methods 3 and 4 are for further marking the location of the sampling area.

[0084] In packaging method 1, the coating does not cover the sampling area of ​​the reagent strip, and the coating is a rectangular structure with a size of 20 mm×45 mm.

[0085] In packaging method 2, the film size is a rectangular structure with a size of 30mm×65mm. The reagent strip is printed with color except for the observation window. The observation window area is transparent and has a size of 8mm×5mm. The sampling area is a 15mm×15mm square, and the 8mm×10mm area inside it is the breakdown area, which is exposed and in contact with the water-absorbing layer.

[0086] In packaging method 3, the film size is a rectangular structure with a size of 30mm×65mm. The reagent strip is printed with color except for the observation window. The observation window area is transparent and has a size of 8mm×6mm. The sampling area is a 20mm×20mm square, and the 18mm×18mm area inside it is the breakdown area, which is exposed and in contact with the water absorption layer. The rectangular film has a perforated reaction area, and the rectangular film also includes a shadowed part, which is the reaction area and the recovery area. The rectangular film also includes a light-transmitting part, that is, the observation area 8mm×6mm.

[0087] In packaging method 4, the film size is a rectangular structure with a size of 30mm×65mm. The reagent strip is printed with color except for the observation window. The observation window area is transparent and has a size of 8mm×6mm. The sampling area is a circle with a diameter of 20mm, and the circular area with an inner diameter of 18mm is the breakdown area, which is exposed and in contact with the water-absorbing layer. The rectangular film has a perforated reaction area. There is also a shadow area on the rectangular film, which is the reaction area and the recovery area. The rectangular film also includes a light-transmitting part, that is, the 8mm×6mm observation area)

[0088] Different volumes of buffer were used to test different packaging methods. The volumes were 20, 40, 60, 80, 100, 120, 150, 200, 300, 500, and 1000 μL. The samples were directly added to the sampling area during the test, and the results were observed after 15 minutes. The test results are shown in Table 6 below:

[0089] Table 6. Test results of different sample volumes for different coating methods

[0090]

[0091] Result analysis: In the above test, when the sample volume in the sampling area of ​​the test groups of packaging methods 1, 3, and 4 was ≥120μL, the test could be carried out normally, the quality control color faded, there were no lines in the test area, and the result was negative; when the sample volume in the sampling area was <120μL, the chromatography failed to be completed or there was a probability of failure to complete the chromatography, the test area still maintained a purple line, and the result was invalid.

[0092] In the above test, when the sampling area of ​​the test group of packaging method 2 is ≥500μL, the test can be carried out normally, the quality control color fades, there is no line in the test area, and the result is negative; when the sample volume in the sampling area is <500μL, the chromatography cannot be completed, the test area still maintains purple lines, and the result is invalid.

[0093] The main reason for the above results is that most of the surface of packaging method 2 is waterproof material. Therefore, when a large amount of liquid is sampled, the liquid flows through the surface layer and disperses on the surface of the waterproof material. Due to the limited speed of liquid absorption in the sampling area, the excess liquid is further recovered by the surface layer, causing the sampling area of ​​the reagent strip to be unable to obtain sufficient sample volume and chromatography failure, and the surface of the pad is wetted in a large area. When the edge of the sampling area contacts the water-absorbing layer (packaging methods 1, 3, and 4), the liquid is more likely to meet the sample volume requirements of the sampling area. The liquid is preferentially absorbed by the sampling area from top to bottom, and the excess liquid is absorbed by the water-absorbing layer through the edge, and it is not easy for the surface of the pad to form a large-area infiltration phenomenon.

[0094] Based on the above results and combined with the appearance of the reagent and user experience, the packaging methods of types 1, 3, and 4 are selected, that is, the exposed sampling area, edge and water absorption layer results. The sampling area indicator patterns and hollow areas include but are not limited to circles, squares, triangles, etc.

[0095] Embodiment 5:

[0096] like Figure 4 As shown, the convenient test strip for premature rupture of membranes in this embodiment is negative:

[0097] Negative result: No lines, indicating no amniotic fluid was detected. If there is any abnormality, continue to monitor.

[0098] Result explanation: If the outflow fluid is only urine sample and does not contain amniotic fluid, only the quality control dye will be recovered by the recovery area due to chromatography, the purple line will fade, and the result will be negative.

[0099] like Figure 5 As shown, the convenient test strip for premature rupture of membranes in this embodiment is positive:

[0100] Positive result: The test line is blue, indicating that amniotic fluid has been detected. It is recommended to seek medical attention in time.

[0101] Result explanation: If the effluent contains amniotic fluid, it contains sufficient IGFBP-1 protein to bind to the latex-labeled anti-human IGFBP-1 monoclonal antibody-2 in the reaction zone, and is captured by the anti-human IGFBP-1 monoclonal antibody-1 in the observation zone during chromatography. The detection line forms a visible blue line. At the same time, the quality control dye is recovered in the recovery zone during chromatography, the purple line fades, and the result is positive.

[0102] like Figure 6 As shown, the convenient test strip for premature rupture of membranes in this embodiment is an invalid result:

[0103] Invalid result: The test line is still purple, indicating insufficient liquid and unsuccessful test. Continue monitoring.

[0104] Result explanation: If there is no obvious outflow or the outflow is insufficient, such as when only vaginal secretions are present, the chromatography cannot be performed, the quality control dye still exists on the detection line of the observation area, and the result is invalid.

[0105] Embodiment 6:

[0106] Clinical sample testing

[0107] The product of the present invention was used in clinical sample testing research. 108 clinically healthy pregnant women (control group) and 48 cases of suspected premature rupture of membranes (observation group) were selected and monitored with the pad. The pad was worn for 5 minutes to 4 hours. When the vaginal discharge is increased or vaginal fluid is discharged, the pad was removed and left for 5 to 15 minutes to observe the results. If there is no abnormality, the pad was removed after 4 hours of wearing and the results were observed directly. If the result is invalid, the pad was replaced and monitoring continued. The monitoring process continued until before delivery. The results are shown in Table 7:

[0108] Table 7. Clinical sample monitoring results

[0109]

[0110] Table 8. Data processing

[0111] Performance Indicators Value (95% CI) Sensitivity 90.476％(69.623％-98.825％) Specificity 98.519％(94.751％-99.820％) Accuracy 97.436％(93.565％-99.297％) Negative predictive value 98.519％(94.679％-99.599％) Positive predictive value 90.476％(70.447％-97.427％)

[0112] Results: According to the results, there was 1 positive sample (1 / 108) and 107 negative samples (107 / 108) in the control group, and no premature rupture of membranes occurred; there were 21 positive samples (21 / 48) in the observation group, of which 19 had premature rupture of membranes (19 / 21), and 2 had normal delivery (2 / 21). At the same time, there were 27 negative samples, of which 2 had premature rupture of membranes (2 / 27), and the remaining 25 samples were all normal delivery. Through data analysis, the product sensitivity was 90.476% (69.623%-98.825%), the specificity was 98.519% (94.751%-99.820%), and the accuracy was 97.436% (93.565%-99.297%). The overall performance was consistent with the clinical diagnosis, and it can be used as an auxiliary diagnostic measure for suspected samples.

[0113] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. .

Claims

1. A device for non-invasive and continuous monitoring of premature rupture of membranes, characterized in that: The invention comprises a reagent strip (10) and a protective pad (20), wherein the protective pad (20) is composed of a surface layer (21), a water absorbing layer (22), and an isolation layer (23) from top to bottom, and the reagent strip (10) is placed between the surface layer (21) and the water absorbing layer (22) along the length direction of the protective pad (20). The reagent strip (10) is sequentially adhered to four functional areas along the chromatography direction: a sampling area (11), a reaction area (12), an observation area (13), and a recovery area (14); The sampling area (11) is a sample pad; The reaction area (12) is a binding pad: anti-human IGFBP-1 monoclonal antibody 2-latex microsphere complex is dried; The observation area (13) is provided with a nitrocellulose membrane, the nitrocellulose membrane is fixed with a detection line (131), and the detection line (131) is composed of an anti-human IGFBP-1 monoclonal antibody 1, a buffer system and a quality control dye; The recovery area (14) is absorbent paper.

2. The device for non-invasive continuous monitoring of premature rupture of membranes according to claim 1, characterized in that: The bottom of the reagent strip (10) is provided with a self-adhesive bottom plate, the outer periphery of the reagent strip (10) is wrapped with waterproof material, and the size of the reagent strip (10) is 40*5-50*10mm; preferably, the size of the reagent strip (10) is 45*8mm; preferably, the waterproof material is a transparent polyolefin film; more preferably, the absorbent paper material includes pulp and fluff pulp. .

3. The device for non-invasive continuous monitoring of premature rupture of membranes according to claim 1, characterized in that: The sample pad is soaked in a diluent and dried, and the diluent includes a buffer system, a blocking protein, and a surfactant.

4. A method for preparing a non-invasive and continuous monitoring device for premature rupture of membranes according to any one of claims 1 to 3, characterized in that: The following steps are included: 1) Cutting and pasting of absorbent paper, nitrocellulose membrane, conjugate pad and sample pad: 2) Preparation, coating and drying of test lines; 3) Preparation, coating and drying of antibody-latex microsphere complexes; 4) Sample pad solution preparation, soaking and drying; 5) Bonding of absorbent paper, nitrocellulose membrane, conjugate pad and sample pad; 6) Cutting, laminating, assembling and packaging.

5. The preparation method according to claim 4, characterized in that: The absorbent paper is cut into 1-1.2*30cm / strip, the nitrocellulose membrane is cut into 1-1.2*30cm / strip, the conjugate pad is cut into 0.8-1*30cm / strip, and the sample pad is cut into 2.5*30cm / strip.

6. The preparation method according to claim 4, characterized in that: In the step 2), PBS is used to dilute the anti-human IGFBP-1 monoclonal antibody to 0.5 to 2 mg / mL, and a quality control dye is added to a final concentration of 0.025 to 0.05 mg / mL, and coated on a nitrocellulose membrane at 1.0 to 2.0 μL / cm, and dried at 50° C. overnight for use.

7. The preparation method according to claim 6, characterized in that: The quality control dye is any one or any combination of rhodamine B, methyl red, phenol red, cresol red and neutral red.

8. The preparation method according to claim 4, characterized in that: The step 3) also includes that the activating agent is 1-ethyl-3[3-dimethylaminopropyl]carbodiimide hydrochloride.

9. The preparation method according to claim 4, characterized in that: The covering film covers the peripheral area of ​​the sample pad, and the peripheral edge of the sampling area is superimposed on the water absorption layer as a whole.

10. The preparation method according to claim 9, characterized in that: The sampling area is a circle with a diameter ≥18 mm or a square with a side length ≥18 mm; preferably, the sampling area is a square with a size of 18 mm*18 mm, or a circle with a diameter of 18 mm.

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