CN103175959B - A kind of preparation method detecting premature rupture of fetal membranes immune chromatography test paper - Google Patents

Abstract

The invention discloses a preparation method of immunochromatographic test paper, in particular to a preparation method of immunochromatographic test paper for detecting premature rupture of membranes. The colloidal gold binding pad in the preparation method is prepared according to the following method: After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to the colloidal gold for 20-50min, adding bovine serum albumin to block for 20- After 50 minutes of centrifugation, the obtained centrifuged product is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in a phosphate buffer solution with a pH of 7.2-7.4, and the phosphate buffer solution contains 10-30mg/100mL of azide Sodium; the phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38° C. for 8-12 hours to obtain the colloidal gold binding pad; The sensitivity and specificity of the chromatography test paper have been significantly improved, reaching over 97% and 93% respectively, thereby reducing the incidence of misdiagnosis.

Description

A preparation method of immunochromatographic test paper for detecting premature rupture of membranes

technical field

The invention relates to a preparation method of immunochromatographic test paper, in particular to a preparation method of immunochromatographic test paper for detecting premature rupture of membranes.

Background technique

Immunochromatography test paper is a method of using the chromatographic principle to flow the target detection molecules or components in the liquid to be tested to the test line (T line) and quality control line (C line) on the test strip. When the test line changes color The change indicates that the test result is positive, otherwise it is negative. When the color of the quality control line changes, it means that the test result is valid, otherwise it is invalid.

Immunochromatographic test strips are very convenient in detecting or diagnosing certain diseases, so they have been fully used in the detection or diagnosis of various diseases or symptoms, such as early pregnancy test strips. In general, immunochromatographic test paper includes the following components: sample pad, colloidal gold binding pad, chromatographic membrane, water-absorbing pad, rubber plate, and the sample pad, colloidal gold-binding pad, chromatographic membrane and water-absorbing pad are sequentially Paste on the rubber plate; the chromatographic membrane is provided with a detection line and a quality control line in sequence; its working principle is: the liquid to be tested is added dropwise to the sample pad, and the liquid to be tested flows to the colloidal gold binding pad using the principle of chromatography. The liquid to be tested dissolves the colloidal gold conjugate in the colloidal gold binding pad, and flows together with it to the detection line and the quality control line on the chromatographic membrane in sequence; when the liquid to be tested contains target detection molecules, the target detection molecules and colloidal gold bind The substance will combine with the antibody components contained in the test line to form an immune complex, and the color of the test line will change at the same time; when the test solution flows to the quality control line, the antibody components contained in the quality control line will combine with the colloidal gold conjugate , so that the color of the quality control line changes.

Rupture of fetal membranes (Rupture of Fetal Membrane, referred to as ROM) may occur at any time during pregnancy, and rupture of fetal membranes that occurs before labor is called premature rupture of membranes (PROM). The incidence of PROM after term (after 37 weeks of gestation) is about 10%, and the incidence of PROM before term (before 37 weeks of gestation) is 2-3.5%. Premature rupture of membranes is the main factor causing prenatal and postpartum complications in obstetric patients, and it is also the main reason for premature birth and neonatal admission to the intensive care unit. Because medical personnel have to balance the prolongation of gestational weeks with the risk of intrauterine and maternal infection and fetal lung development problems, the management of patients with premature rupture of membranes is costly and difficult. Accurate and timely diagnosis of premature rupture of membranes in pregnant women Breaking is crucial.

Among the existing methods for detecting premature rupture of membranes, in addition to previous methods such as clinical history inquiry, the detection tool mainly uses intercellular adhesion molecule-1 (ICAM-1) in the vaginal secretions of pregnant women as the detection index. new method. Among them, the immunochromatographic test paper for detecting premature rupture of membranes with ICAM-1 as the detection index has played a good effect on the timely diagnosis of whether premature rupture of membranes occurs for obstetric patients; the detection line of the immunochromatographic test paper is coated with sheep antibody Human ICAM-1 polyclonal antibody working solution, the quality control line is coated with goat anti-mouse IgG working solution, and the colloidal gold conjugate contains mouse anti-human ICAM-1 monoclonal antibody.

Although the existing immunochromatographic test paper for detecting premature rupture of membranes using sICAM-1 as a detection index provides convenience for obstetric patients, its sensitivity, specificity and accuracy are not ideal, which easily leads to misdiagnosis and is harmful to obstetric patients. It will also cause inconvenience.

Contents of the invention

In view of this, the present invention provides a method for preparing immunochromatographic test paper for detecting premature rupture of membranes. The sensitivity and specificity of the immunochromatographic test paper for detecting premature rupture of membranes prepared by the preparation method are significantly improved, respectively. It can reach more than 97% and more than 93%, thereby reducing the incidence of misdiagnosis.

In order to solve the above technical problems, the technical solution of the present invention adopts a preparation method for detecting premature rupture of membranes immunochromatographic test paper, and the preparation method includes the following steps:

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to the colloidal gold for 20-50min labeling, adding bovine serum albumin and centrifuging after blocking for 20-50min, the obtained centrifugal product is a colloidal gold conjugate; The obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 10-30mg/100mL is contained in the described phosphate buffer; Dry on glass fiber for 8-12 hours at 36-38°C to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper.

Preferably, in the step B, the phosphate buffer contains 20 mg/100 mL of sodium azide.

Preferably, the ratio of the amount of the mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody added in the step B to the colloidal gold is 150-180 μg/mL.

Preferably, the ratio of the amount of bovine serum albumin added in the step B to the colloidal gold is 150-200mg/100mL.

Preferably, in the step A, the concentration of the detection line working solution is 1.5-2.5 mg/mL, and the concentration of the quality control line working solution is 0.5-1.5 mg/mL; Dry for 8-12 hours to obtain a chromatographic membrane.

Preferably, in the step A, the concentration of the detection line working solution is 2.5 mg/mL, and the concentration of the quality control line working solution is 1.5 mg/mL.

Preferably, the coated nitrocellulose membrane in the step A is dried at 38° C. for 10 hours.

Preferably, the sample pad in step C is prepared by the following method: soak the glass fiber in a phosphate buffer solution with a pH of 7.2-7.4 for 2-6 hours, and then dry it at 36-38°C for 8-12 hours Get a sample pad.

Preferably, in step C, the phosphate buffer also contains 0.5-3.0 mL/100 mL of Triton-X100 and 0.5-2 g/100 mL of BSA.

Compared with the prior art, the present invention is described in detail as follows:

The technical scheme adopted in the present invention is: the preparation method of detecting premature rupture of membranes immunochromatographic test paper, and described preparation method comprises the following steps:

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to the colloidal gold for 20-50min labeling, adding bovine serum albumin and centrifuging after blocking for 20-50min, the obtained centrifugal product is a colloidal gold conjugate; The obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 10-30mg/100mL is contained in the described phosphate buffer; Dry on glass fiber for 8-12 hours at 36-38°C to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper.

First of all, intercellular adhesive molecule 1 (ICAM-1) is a general term for molecules involved in the interaction between cells and between cells and extracellular matrix, including soluble intercellular adhesion molecules present in body fluids. Adhesion molecule (soluble intercellular adhesive molecule 1, referred to as sICAM-1) and membrane-type intercellular adhesion molecule (abbreviated as membrane-type ICAM-1) present on the cell surface. Among them, sICAM-1 not only retains the biological characteristics of membrane-type ICAM-1, but also is a soluble form of membrane-type ICAM-1 in body fluids. The inventors have found through sufficient research that pregnant women with premature rupture of membranes are affected by the expression of ICAM-1 on monocytes in the fetal membranes and amniotic fluid and the influence of neutrophils, and the ICAM-1 content in their vaginal secretions Significantly increased, including the content of membrane-type ICAM-1 and sICAM-1. There are existing detection tools that use ICAM-1 as the target molecule to detect premature rupture of membranes, but in the actual application process, its sensitivity and specificity are low, and it is easy to cause misdiagnosis.

After sufficient research, the inventor found that the cause of the increase of ICAM-1 content in the vaginal secretions of pregnant women with premature rupture of membranes is mainly due to the change of sICAM-1 content, rather than the membranous type with less change in content. ICAM-1; the inventors have found through experiments that sICAM-1 is one of the main proteins that appear in amniotic fluid in the third trimester of pregnancy, and its content is generally about 14 ng/mL; when premature rupture of membranes occurs, except for the Membrane-type ICAM-1 will flow out of the vagina with amniotic fluid, mainly in amniotic fluid, while sICAM-1, which is more abundant in amniotic fluid, will appear in vaginal secretions. Therefore, using sICAM-1 in the vaginal secretions of pregnant women as the detection target molecule may have better sensitivity and specificity than using ICAM-1 as the detection target molecule to detect premature rupture of membranes.

In view of the above-mentioned experimental results and the sufficient research of the inventors, in the preparation method of the immunochromatographic test paper for detecting premature rupture of membranes described in the technical scheme of the present invention, goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the detection line Working solution, goat anti-mouse IgG polyclonal antibody as quality control line working solution.

The sensitivity of immunochromatographic test strips when used to detect diseases is the percentage of positive samples in the sick population to the total number of patients, and the specificity is the percentage of negative samples in the healthy population to the total number of healthy people. There are many factors that affect the sensitivity and specificity, including the difference in the manufacturing process of the test paper itself and whether the personal operation is standardized, etc., but basically the manufacturing process of the test paper itself is the main factor. The manufacturing process of immunochromatographic test paper includes several key steps, mainly the preparation of chromatographic membrane, colloidal gold, colloidal gold binding pad and sample pad.

The present invention finds through the protein chip screening test that compared with the confirmed women with premature bankrupt membranes and non-premature bankrupt women (the age of the two groups of puerperas matches the gestational age, no underlying disease, no pregnancy complications), the The concentration of sICAM-1 in the vaginal secretion samples was significantly different; among them, more than 90% of the sICAM-1 concentrations in the vaginal secretion samples of women with confirmed premature bankrupt membranes were above 2ng/mL, while the confirmed non-premature membranes More than 90% of the concentration of sICAM-1 in the vaginal secretion samples of bankrupt women were below 1 ng/mL.

In view of the above experimental results, in order to allow the immunochromatographic test paper to correctly detect whether premature rupture of membranes occurs in pregnant women, the preparation method of the immunochromatographic test paper needs to be adjusted to a certain extent. The preparation method of the colloidal gold conjugate in order to improve the sensitivity and specificity of the immunochromatographic test paper.

The colloidal gold conjugate of the present invention adopts the above-mentioned preparation method, and while the preservation time of the obtained colloidal gold conjugate is relatively ideal, the chromatographic effect of the colloidal gold binding pad prepared by it is also comparatively ideal, thereby the sensitivity and specificity of the obtained immunochromatographic test paper Performance can be significantly improved.

The colloidal gold conjugate of the present invention is a conjugate of mouse anti-human soluble intercellular adhesion molecule monoclonal antibody and colloidal gold. In the existing preparation methods of colloidal gold conjugates, sodium chloride solution is usually added to prolong its shelf life, and sodium azide is added to the phosphate buffer solution to better extend the shelf life. Although the addition of sodium chloride and sodium azide will prolong the shelf life of the colloidal gold conjugate, the addition of the two will reduce the chromatographic ability of the resulting colloidal gold binding pad to a certain extent, thereby reducing the sensitivity of the immunochromatographic test paper and specificity.

In the preparation method of the colloidal gold conjugate of the present invention, no sodium chloride solution is added, and the concentration of sodium azide is adjusted to 10-30mg/100mL. This preparation condition has little influence on the chromatographic ability of the obtained colloidal gold binding pad, and the obtained The sensitivity and specificity of the immunochromatographic test paper are effectively improved, and at the same time, the shelf life of the obtained colloidal gold conjugate will not be affected.

Further, the present invention preferably adopts sodium azide containing 20mg/100mL in the phosphate buffer. In the present invention, on the basis of the above preparation conditions, the concentration of sodium azide in the phosphate buffer can be further adjusted, specifically preferably 20 mg/mL.

Further, in the present invention, the ratio of the amount of the added mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to the colloidal gold is preferably 150-180 μg/100 mL. In the preparation process of the colloidal gold conjugate of the present invention, it can be further preferred that the amount of the mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody accounts for 150-180 mg/100 mL of the colloidal gold. In the preferred embodiment The chromatographic ability of the obtained colloidal gold binding pad is better, and the sensitivity and specificity of the obtained immunochromatographic test paper are improved to a certain extent. Furthermore, in the present invention, the ratio of the added bovine serum albumin to the colloidal gold is preferably 150-200mg/100mL.

Further, the present invention preferably uses the sample pad in step C to be prepared by the following method: soak the glass fiber in a phosphate buffer solution with a pH of 7.2-7.4 for 2-6 hours, and then dry it at 36-38°C 8-12 hours to get a sample pad. The sample pad of the present invention can adopt existing known preparation method to prepare gained, also can adopt to buy commercially available product; The present invention preferably adopts described sample pad to adopt above-mentioned preferred method to prepare gained, and the obtained sample pad of this method is for the obtained test paper The sensitivity and specificity can be improved to a certain extent.

Further, in the step A, the concentration of the detection line working solution is 1.5-2.5 mg/mL, and the concentration of the quality control line working solution is 0.5-1.5 mg/mL; Dry for 8-12 hours to obtain a chromatographic membrane.

The different concentrations of the antibody in the detection line (in the present invention, goat anti-human soluble intercellular adhesion molecule polyclonal antibody) have significant effects on the antigenic components in the detection sample (in the present invention, it is the soluble intercellular adhesion molecule in vaginal secretions of pregnant women). ) have different effects; when the concentration of the antibody in the test line is too low, the amount of the antibody that reacts with the antigen is small, resulting in inconspicuous color development, resulting in a positive result that should have been detected in the sick population Negative results are detected, and the sensitivity is low; when the concentration of the antibody in the detection line is too high, the amount of its binding reaction with the antigen is large, resulting in too obvious color development, resulting in negative results that should have been detected in healthy people Positive results were detected with low specificity.

The different concentrations of antibodies in the quality control line (goat anti-mouse IgG polyclonal antibody in the present invention) have different effects on the determination of the validity of the test results of the test paper; The antibody in the pad (mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in this invention) will combine with the antibody in the detection line to form a conjugate with a certain color, and some colloidal gold binding pads that are not bound The antibody in the quality control line will continue to move to the quality control line, so as to combine with the antibody components in the quality control line to form another conjugate, and the color of the quality control line will change, thus indicating that the test result is valid. When the concentration of the working solution of the detection line is too high, the antibody content of the colloidal gold binding pad moving to the quality control line will be less, resulting in the color change of the quality control line is not obvious enough, resulting in invalid test results; when the working solution of the detection line If the concentration is low, the antibody content of the colloidal gold binding pad moving to the quality control line will be higher, resulting in a waste of the cost of the working solution of the quality control line.

In view of the above reasons, if only the concentration of the working solution of the detection line and the concentration of the working solution of the quality control line are adjusted separately, the optimal concentration when the two are used alone is obtained, but since the detection line and the quality control line in the immunochromatography test strip are Therefore, the optimal concentration after the combination of the two needs to be screened and verified through a large number of experiments. The inventor found through sufficient research that on the basis of the existing method for preparing and detecting premature rupture of membranes immunochromatographic test paper, the concentration of the working solution of the detection line and the concentration of the working solution of the quality control line were adjusted to 1.5-2.5 mg/mL and 1.5 mg/mL respectively. When the concentration is 0.5-1.5mg/mL, the obtained test paper can have better sensitivity and specificity under the same usage method.

In addition, the nitrocellulose membrane coated with the detection line and quality control line obtained in the preparation method of the immunochromatographic test paper for detecting premature rupture of membranes of the present invention needs to be dried at 36-38° C. for 8-12 hours to obtain a chromatographic membrane. After the coated nitrocellulose membrane is dried at a certain temperature, the sensitivity and specificity of the test paper can also be improved to a certain extent.

Further, the present invention preferably adopts the concentration of the working solution of the detection line in the step A as 2.5 mg/mL, and the concentration of the working solution of the quality control line as 1.5 mg/mL. In the preparation method of the present invention, the concentration of the working solution of the detection line is 1.5-2.5 mg/mL, and the concentration of the working solution of the quality control line is 0.5-1.5 mg/mL; the present invention has been screened and tested in the above-mentioned concentration range, and is preferred in the preparation method of the present invention. It is 2.5mg/mL to adopt the detection line working solution concentration, and the quality control line working solution concentration is 1.5mg/mL. The present invention preferably adopts the combination of the above two concentrations. Sensitivity and specificity were significantly improved.

Further, in the present invention, the coated nitrocellulose membrane in the step A is preferably dried at 38°C for 10 hours. In the preparation method of the present invention, the coated nitrocellulose membrane is dried at 36-38° C. for 8-12 hours, and the present invention preferably adopts drying at 38° C. for 10 hours.

Further, the present invention preferably uses the sample pad in step C to be prepared by the following method: soak the glass fiber in a phosphate buffer solution with a pH of 7.2-7.4 for 2-6 hours, and then dry it at 36-38°C 8-12 hours to get a sample pad. The sample pad of the present invention can adopt existing known preparation method to prepare gained, also can adopt to buy commercially available product; The present invention preferably adopts described sample pad to adopt above-mentioned preferred method to prepare gained, and the obtained sample pad of this method is for the obtained test paper The sensitivity and specificity can be improved to a certain extent.

Further, in the present invention, it is preferred that the phosphate buffer in step C further contains 0.5-3.0 mL/100 mL of Triton-X100 and 0.5-2 g/100 mL of BSA. Triton-X100 of the present invention is polyethylene glycol octyl phenyl ether, which is a surfactant; BSA is bovine serum albumin; the present invention adopts the above-mentioned two substances to be jointly prepared in phosphate buffer saline And it is used to soak glass fiber, and the sample pad prepared by it has a better buffering effect on the sample to be tested, so that the sensitivity and specificity of the test paper are obviously improved.

Description of drawings

Figure 1 is a diagram of the detection results of the cytokine/chemokine antibody chip of the vaginal secretion sample in the premature rupture of membranes group;

Figure 2 is a diagram of the results of the cytokine/chemokine antibody chip detection of the vaginal secretion samples of the healthy control group;

Figure 3 is a diagram of the detection results of the cytokine/chemokine antibody chip of amniotic fluid samples from healthy pregnant women;

Fig. 4 is a graph showing the detection results of sICAM-1 by enzyme-linked immunosorbent assay.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

Example 1

Premature rupture of membranes group: 110 pregnant women with premature rupture of membranes (including 80 cases of full-term premature rupture of membranes and 30 cases of preterm premature rupture of membranes);

Healthy control group: 110 pregnant women with intact membranes before delivery;

Amniotic fluid sample group: 30 pregnant women with intact membranes undergoing cesarean section.

Clinical screening criteria for experimental subjects:

Premature rupture of membranes group—(i) Leakage of amniotic fluid was observed before labor;

(ii) The pH test paper of the vaginal secretion sample is positive;

(iii) Positive test for fern leaf crystals.

Healthy control group—(i) Amniotic fluid leakage was not observed before labor;

(ii) The pH test paper test of the vaginal secretion sample is negative;

(iii) Negative test for fern leaf crystals.

The age distribution of the experimental subjects:

Sample collection method: Pregnant women take the bladder lithotomy position (lying in the supine position, with the lower limbs flexed separately, exposing the vulva), peeking through the vagina, inserting a disposable sterile cotton swab into the posterior fornix of the vagina, and rotating it 5 times to sample . Insert the cotton swab head into 1mL sample diluent (phosphate buffer system), rotate 5 times, squeeze and rotate 2 times against the tube wall to obtain the sample.

A. Experimental method: qualitative detection of cytokine/chemokine antibody chip

Experimental conditions:

1) Put the membrane (RayBiotech, USA) pre-labeled with 174 cytokine antibodies into the detection box, then add 2ml of blocking solution, and block at room temperature for 6 hours;

2) Discard the blocking solution, add 1.2ml of the sample to be tested, and incubate overnight at 4°C;

3) Discard the sample and wash 5 times with 2ml washing buffer;

4) Add 1ml biotin-conjugated detection antibody and incubate at room temperature for 2 hours;

5) Discard the antibody and wash 5 times with 2ml washing buffer;

6) Add 2ml horseradish peroxidase (HRP)-coupled streptavidin and incubate at room temperature for 2 hours;

7) Discard the liquid and wash 5 times with 2ml washing buffer;

8) Add 500 μl of chemiluminescent reagent to the membrane, and keep it in the dark for 2 minutes, and observe the result.

The samples to be tested collected from the premature rupture of membranes group, the healthy control group and the amniotic fluid sample group were all tested under the above experimental conditions, and the obtained results are shown in Fig. 1 , Fig. 2 and Fig. 3 .

Experimental results: Mark 1 in Figure 1, Figure 2 and Figure 3 is the expression signal of IGFBP-1, Mark 2 is the expression signal of sICAM-1, and Mark 3 is the expression signal of Axl. It can be seen from Figure 1, Figure 2 and Figure 3 that the expressions of IGFBP-1, sICAM-1, and Axl in the vaginal secretions and amniotic fluid samples of the premature rupture of membranes group were stronger, while the vaginal secretions of healthy pregnant women Among the above three, the expression of the above three was weak, and among the above three, the expression of sICAM-1 was the strongest.

B. Experimental method: quantitative detection by enzyme-linked immunosorbent assay

Experimental conditions:

Experimental material: sICAM-1ELISA detection kit (R&D company)

1. Preparation of reagents

Wash Buffer

Substrate Solution: Before use, mix equal amounts of chromogenic reagent A and chromogenic reagent B in the kit for 15 minutes in the dark, and each well requires 200 μl of the mixture of the two chromogenic reagents.

sICAM-1 standard: Dilute the sICAM-1 standard with 1 ml of deionized water. The concentration of the diluted standard stock solution is 250ng/ml. To ensure that the standard is thoroughly mixed, shake the standard on a shaker for at least 15 minutes before further dilution.

The 250ng/ml standard was prepared as the sICAM-1 detection standard with concentrations of 50ng/ml, 25ng/ml, 12.5ng/ml, 6.25ng/ml, 3.13ng/ml, and 1.56ng/ml; RD5-7 Calibration diluent as blank control (0ng/ml)

2. Detection procedure

Add 100 μl of sICAM-1 Conjugate to each microwell; add 100 μl of standard, control and sample into corresponding microwells. Cover the wells with the gel strips provided in the kit. The microplate was incubated on a horizontal shaker at 500±50 rpm for 1.5 hours at room temperature. Wash the plate; add 200 μl of substrate solution (Substrate Solution) to each well, and incubate for 30 minutes in the dark at room temperature; add 50 μl of stop solution (Stop Solution) to each well. The color of the solution in the wells will change from blue to yellow. If the color of the solution in the well is green or the color changes are inconsistent, gently tap the plate to ensure that the solution is fully mixed; measure the absorbance at 450nm within 30 minutes.

3. Make a standard curve and calculate the sample content.

4. The calculated content of each sample to be tested is counted and drawn into a chart, and the resulting chart is shown in Figure 4.

Experimental results: In Figure 4, AF of control is the amniotic fluid sample, CVF of PROM is the vaginal secretion of the premature rupture of membranes group, and CVF of control is the vaginal secretion of the healthy control group. It can be seen from Figure 4 that the concentration of sICAM-1 in the amniotic fluid samples of healthy pregnant women is the highest, reaching an average of 80 ng/mL; the concentration of sICAM-1 in the vaginal secretion samples of the premature rupture of membranes group is more than 90% and can reach 2 ng More than 90% of the concentration of sICAM-1 in the vaginal secretion samples of the healthy control group was below 1 ng/mL.

From the experimental results of Example 1, it can be seen that one of the main content changes in the vaginal secretions of pregnant women with premature rupture of membranes is soluble intercellular adhesion molecule sICAM-1, and sICAM-1 mainly comes from the rupture of membranes. amniotic fluid.

The following is a control example of the immunochromatographic test paper prepared according to different preparation methods using sICAM-1 in the vaginal secretions of pregnant women as the target molecule for detection. The preparation methods of each control example are as follows:

Comparative example 1

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, then add 10g/100mL sodium chloride, and centrifuge. Gained centrifugal product is colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffered saline solution of pH7.2-7.4, contains the sodium azide of 90mg/100mL in the described phosphate buffered saline solution; Added colloidal gold The phosphate buffer solution of the conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber sheet in order of the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare Comparative Example 1-detection of early fetal membranes Immunochromatography test paper is broken; Described sample pad, absorbent pad are all purchased commercially available products.

Comparative example 2

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, and then add 5g/100mL sodium chloride to obtain the centrifuged product It is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 80mg/100mL is contained in the described phosphate buffer; The phosphate buffer solution is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in order of the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare Comparative Example 2-detection of early fetal membranes Immunochromatography test paper is broken; Described sample pad, absorbent pad are all purchased commercially available products.

Comparative example 3

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, and then add 5g/100mL sodium chloride to obtain the centrifuged product It is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 5mg/100mL is contained in the described phosphate buffer; The phosphate buffer solution is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber sheet in order of the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare Comparative Example 3-detection of early fetal membranes Immunochromatography test paper is broken; Described sample pad, absorbent pad are all purchased commercially available products.

Comparative example 4

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for labeling for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, and then add 1g/100mL sodium chloride to obtain the centrifuged product It is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 10mg/100mL is contained in the described phosphate buffer; The phosphate buffer solution is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in order of the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare Comparative Example 4-detection of early fetal membranes Immunochromatography test paper is broken; Described sample pad, absorbent pad are purchased commercially available product.

Comparative example 5

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, and then add 5g/100mL sodium chloride to obtain the centrifuged product It is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 20mg/100mL is contained in the described phosphate buffer; The phosphate buffer solution is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber sheet in order of the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare Comparative Example 5-detection of fetal membranes Immunochromatography test paper is broken; Described sample pad, absorbent pad are all purchased commercially available products.

Comparative example 6

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, and then add 5g/100mL sodium chloride to obtain the centrifuged product It is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 30mg/100mL is contained in the described phosphate buffer; The phosphate buffer solution is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 7

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to colloidal gold for labeling for 20-50 minutes, add bovine serum albumin to block for 20-50 minutes, and then add 1g/100mL sodium chloride to obtain the centrifuged product It is a colloidal gold conjugate; the obtained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 25mg/100mL is contained in the described phosphate buffer; The phosphate buffer solution is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain a colloidal gold bonding pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 8

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 150 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 9

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 180 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 150 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 10

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, The concentration of the working solution of the quality control line is 1-3 mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative Example 11

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 2mg/mL, and the quality control The concentration of the line working solution is 2 mg/mL, and the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 12

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 3 mg/mL, and the quality control The concentration of the line working solution is 3mg/mL, and the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 13

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 1.5mg/mL, and the quality The concentration of the control line working solution is 0.5mg/mL, the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane, and the coated nitrocellulose membrane is dried at 36-38°C 8-12 hours to get the chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative example 14

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 1.5mg/mL, and the quality The concentration of the control line working solution is 1.5mg/mL, the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane, and the coated nitrocellulose membrane is dried at 36-38°C 8-12 hours to get the chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative Example 15

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 2.5mg/mL, and the quality The concentration of the control line working solution is 0.5mg/mL, the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane, and the coated nitrocellulose membrane is dried at 36-38°C 8-12 hours to get the chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative Example 16

A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 2.5mg/mL, and the quality The concentration of the control line working solution is 1.5mg/mL, the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane, and the coated nitrocellulose membrane is dried at 38°C for 10 hours get chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatographic test paper; the sample pad and the absorbent pad are commercially available products.

Comparative Example 17

A. Goat anti-human soluble intercellular adhesion molecule-1 is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, and the quality control line The concentration of the working solution is 1-3mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper; the sample pad is prepared by the following method: soak the glass fiber in the phosphate buffer solution of pH 7.2-7.4 for 2 hours, and then dry the sample pad at 36°C for 8 hours; the absorbent pad For purchasing commercially available products.

Comparative Example 18

A. Goat anti-human soluble intercellular adhesion molecule-1 is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, and the quality control line The concentration of the working solution is 1-3mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper; the sample pad is prepared by the following method: soak the glass fiber in a phosphate buffer solution with a pH of 7.2-7.4 for 6 hours, and then dry it at 38°C for 12 hours to obtain a sample pad; the absorbent pad For purchasing commercially available products.

Comparative Example 19

A. Goat anti-human soluble intercellular adhesion molecule-1 is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, and the quality control line The concentration of the working solution is 1-3mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper; the sample pad is prepared by the following method: soak the glass fiber in the phosphate buffer solution of pH 7.2-7.4 for 2-6 hours, and then dry it at 36-38°C for 8-12 hours to obtain the sample pad; the phosphate buffer contains 0.5mL/100mL of Triton-X100 and 0.5g/100mL of BSA; the absorbent pad is a commercially available product.

Comparative example 20

A. Goat anti-human soluble intercellular adhesion molecule-1 is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the detection line working solution is 1-3mg/mL, and the quality control line The concentration of the working solution is 1-3mg/mL, and the working solution of the detection line and the working solution of the quality control line are coated on the nitrocellulose membrane to obtain a chromatographic membrane;

B. Add 150 μg of mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody in proportion to every 100 mL of colloidal gold for labeling for 20-50 min, then add 200 mg of bovine serum albumin in proportion to every 100 mL of colloidal gold for blocking for 20-50 min After centrifugation, the resulting centrifugal product is a colloidal gold conjugate; the resulting colloidal gold conjugate is dissolved in a phosphate buffer of pH 7.2-7.4, and the phosphate buffer contains 20mg/100mL of sodium azide; The phosphate buffer solution added with the colloidal gold conjugate is coated on the glass fiber, and dried at 36-38°C for 8-12 hours to obtain the colloidal gold conjugate pad;

C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper; the sample pad is prepared by the following method: soak the glass fiber in the phosphate buffer solution of pH 7.2-7.4 for 2-6 hours, and then dry it at 36-38°C for 8-12 hours to obtain the sample pad; the phosphate buffer contains 3.0mL/100mL of Triton-X100 and 2g/100mL of BSA; the absorbent pad is a commercially available product.

The sources of material in the above-mentioned comparative examples 1-20 are as follows:

Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody: purchased from R&D Company;

Goat anti-mouse IgG polyclonal antibody: purchased from Shanghai Paikun Bioengineering Co., Ltd.;

Mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody: purchased from R&D Company;

Sample pad (commercially available): purchased from Ahlstrom;

Absorbent pad (commercially available): purchased from Shanghai Jieyi Biotechnology Co., Ltd.;

Colloidal gold (commercially available): purchased from Shanghai Jiening Biotechnology Co., Ltd.;

Bovine serum albumin (BSA): Roche company;

Example 2

The control example 1-20 immunochromatographic test papers prepared by different methods were used to detect the clinical samples of vaginal secretions of the same pregnant women respectively, including premature rupture of membranes group and healthy control group, wherein the premature rupture of membranes group was 240 cases, 260 cases in the healthy control group, and the test subjects were all clinically diagnosed and screened using the method in Example 1.

Sample collection: Pregnant women were taken in the bladder lithotomy position (lying in the supine position, with the lower limbs flexed separately, exposing the vulva), after peeping through the vagina, a disposable sterile cotton swab was inserted into the posterior fornix of the vagina, and rotated 5 times to sample. Insert the cotton swab head into 1mL sample diluent (phosphate buffer system), rotate 5 times, squeeze and rotate 2 times against the tube wall to obtain the sample.

Sample test subject age distribution:

Utilize control example 1-20 to detect respectively to collected 500 routine samples, the negative and positive case number statistics of gained detection result are shown in Table 1:

Table I

In the above table 1, comparative example 1-3 is the immunochromatographic test paper prepared by the existing preparation method; comparative example 4-7 is the preparation method of the colloidal gold conjugate in the colloidal gold bonding pad only adjusted relative to comparative example 1-3. Chromatographic test paper; Comparative example 8-10 is the main preparation method of the present invention; Comparative example 11-20 is the further improved preparation method of the present invention, wherein Comparative example 11-16 mainly further improved the preparation method of colloidal gold conjugate, Comparative example 17-20 mainly further improves the preparation method of the sample pad.

Can draw from the difference of last table 1 data and comparative example, preparation method of the present invention can obviously improve the sensitivity and the specificity of gained immunochromatography test paper; Sensitivity and specificity are also significantly improved.

The above are only preferred implementations of the present invention, and it should be noted that the above preferred implementations should not be regarded as limiting the present invention, and the scope of protection of the present invention should be based on the scope defined in the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (2)

1. a preparation method for detecting premature rupture of membranes immunochromatographic test paper, is characterized in that: described preparation method comprises the following steps: A. Goat anti-human soluble intercellular adhesion molecule-1 polyclonal antibody is used as the working solution of the detection line, and goat anti-mouse IgG polyclonal antibody is used as the working solution of the quality control line; the concentration of the working solution of the detection line is 2.5mg/mL, and the quality The concentration of the control line working solution is 1.5mg/mL, the detection line working solution and the quality control line working solution are coated on the nitrocellulose membrane, and the coated nitrocellulose membrane is dried at 36-38°C 8-12 hours to get the chromatographic membrane; B. After adding mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody to the colloidal gold for 20-50min labeling, adding bovine serum albumin and centrifuging after blocking for 20-50min, the obtained centrifugal product is a colloidal gold conjugate; Gained colloidal gold conjugate is dissolved in the phosphate buffer of pH7.2-7.4, and the sodium azide of 20mg/100mL is contained in the described phosphate buffer; The phosphate buffer that has added colloidal gold conjugate is coated on On the glass fiber, dry at 36-38°C for 8-12 hours to obtain a colloidal gold binding pad; the amount of the added mouse anti-human soluble intercellular adhesion molecule-1 monoclonal antibody accounts for 150-180 μg/ 100mL; the amount of bovine serum albumin added accounts for the ratio of colloidal gold to be 150-200mg/100mL; C. Paste the chromatographic membrane and colloidal gold binding pad obtained in steps A and B on the rubber plate in sequence according to the sample pad, colloidal gold binding pad, chromatographic membrane, and water-absorbing pad to prepare the immune system for detecting premature rupture of membranes. Chromatography test paper; the sample pad is prepared by the following method: soak the glass fiber in the phosphate buffer solution of pH 7.2-7.4 for 2-6 hours, and then dry it at 36-38°C for 8-12 hours to obtain the sample pad; in this step, the phosphate buffer also contains 0.5-3.0mL/100mL Triton-X100 and 0.5-2g/100mL BSA. 2. The preparation method according to claim 1, characterized in that: in the step A, the coated nitrocellulose membrane is dried at 38° C. for 10 hours.