CN118275681A - CA125, CA153 and CEA combined detection immunochromatography test strip, kit and preparation method of test strip - Google Patents
CA125, CA153 and CEA combined detection immunochromatography test strip, kit and preparation method of test strip Download PDFInfo
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Abstract
The application discloses a multi-joint detection immunochromatography test strip, a kit and a preparation method of the test strip, and belongs to the field of in-vitro diagnosis. The test strip comprises a sample pad, a latex pad, an NC film, water absorbing paper and a bottom plate, wherein the sample pad, the latex pad, the NC film and the water absorbing paper are sequentially overlapped on the bottom plate, and the latex pad is coated with a Eu microsphere marked CA125 monoclonal antibody I and a Bodipy fluorine boron microsphere marked CEA monoclonal antibody I; the marked line of the NC film is coated with Alexa Fluor 647 marked CA153 monoclonal antibody I; the detection line of the NC film is coated with a CA125 monoclonal antibody II, a CA153 monoclonal antibody II and a CEA monoclonal antibody II; the NC membrane is coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody on the quality control line. The application also comprises a kit comprising the test strip and a preparation method of the test strip. The test strip provided by the application can be used for simultaneously detecting the CA125, CA153 and CEA tumor markers, so that the degree of difference of diagnosis results when a single tumor marker is detected is reduced, the diagnosis efficiency is improved, and the clinical diagnosis requirement is met.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to a multi-joint detection immunochromatography test strip, a kit and a preparation method of the test strip.
Background
Lung cancer is the leading cause of cancer-related death. Because of the relatively large number of lung cancer types, there are cases of interconversion between different lung cancer types. Non-small cell lung cancer is a common type of lung cancer, and the early stage of the disease has no specific symptoms, often manifests as cough, chest pain, shortness of breath and the like, but often is mistakenly pneumonia or pneumothorax, thereby leading to misdiagnosis. The pathological examination is a gold standard for clinical diagnosis of non-small cell lung cancer, but can cause a certain wound to patients, and has long examination time and limited clinical application range. The tumor markers are biological substances synthesized and released by tumor cells or substances generated by the reaction of organisms on tumor tissues, the tumor marker level can be abnormally changed due to the gene expression of the tumor cells in the process of tumor generation and growth, and the tumors can be judged and identified through the immune characteristics of the tumor markers and can reflect the activity of tumor generation and growth. The CA125, CA153 and CEA tumor markers are sensitive indexes in the early diagnosis of the non-small cell lung cancer, and abnormal changes of the indexes of the non-small cell lung cancer patients can be found by detecting the level of the sensitive indexes, and the abnormal changes do not need to be positioned, are safe in a minimally invasive way, are rapid and accurate, and are easy to popularize.
However, single CA125, CA153, CEA tumor markers have large differences in diagnosis specificity, sensitivity and relativity to different types of lung cancer, in addition, serum detection of CA125, CA153, CEA tumor markers is mostly a chemiluminescent method, only one index can be detected at a time, the detection time is long, the detection cost is high, the real-time monitoring is difficult, and the clinical diagnosis requirement cannot be met.
Disclosure of Invention
The invention discloses a CA125, CA153 and CEA combined detection immunochromatography test strip and a preparation method thereof, which solve the problems of large diagnosis result difference and low detection efficiency in the prior art for detecting single CA125, CA153 and CEA tumor markers.
The invention discloses a CA125, CA153 and CEA combined detection immunochromatographic test strip, which comprises a sample pad, a latex pad, an NC film, water absorbing paper and a bottom plate, wherein the sample pad, the latex pad, the NC film and the water absorbing paper are sequentially overlapped on the bottom plate, and Eu microsphere marked CA125 monoclonal antibody I and Bodipy fluoroboron microsphere marked CEA monoclonal antibody I are coated on the latex pad; the marked line of the NC film is coated with Alexa Fluor 647 marked CA153 monoclonal antibody I; the detection line of the NC film is coated with a CA125 monoclonal antibody II, a CA153 monoclonal antibody II and a CEA monoclonal antibody II; the NC membrane is coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody on the quality control line.
In some embodiments, the Eu microsphere labeled CA125 monoclonal antibody I is obtained by coupling a streptavidin Eu microsphere with a biotin labeled CA125 monoclonal antibody I.
In some embodiments, the Bodipy fluoroboro microsphere-labeled CEA monoclonal antibody I is obtained from a coupling reaction of streptavidin Bodipy fluoroboro microsphere with biotin-labeled CA125 monoclonal antibody I.
In some embodiments, alexa Fluor 647 labeled CA153 monoclonal antibody IAlexa Fluor 647 labeled CA153 monoclonal antibody I is obtained by proportionally mixing Alexa Fluor 647 and CA153 monoclonal antibody I, and dialyzing.
In some embodiments, the coating concentrations of CA125 monoclonal antibody II, CA153 monoclonal antibody II, and CEA monoclonal antibody II on the detection line of the NC membrane are: 2.0mg/mL, 1.5mg/mL and 2.0mg/mL; the NC membrane was coated with 1.0mg/mL of goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody at 0.8uL/cm on the control line.
In a second aspect, the invention also discloses a method for preparing the immunochromatographic test strip of the first aspect, which comprises the following steps: preparing a CA125 monoclonal antibody I marked by Eu microspheres; preparing a Bodipy fluorine boron microsphere marked CEA monoclonal antibody I; preparing Alexa Fluor 647 marked CA153 monoclonal antibody I; coating a Eu microsphere marked CA125 monoclonal antibody I and a Bodipy fluorine boron microsphere marked CEA monoclonal antibody I on a latex pad; coating an Alexa Fluor 647 marked CA153 monoclonal antibody I on a marked line of an NC film, coating a CA125 monoclonal antibody II, a CA153 monoclonal antibody II and a CEA monoclonal antibody II on a detection line of the NC film in sequence, and coating a goat anti-mouse IgG antibody or a rabbit anti-mouse IgG antibody on a quality control line of the NC film; the sample pad, latex pad, NC film and water absorbing paper prepared in this order were lapped and fixed on the base plate.
In a third aspect, the invention also discloses a combined detection immunochromatographic test strip containing the CA125, CA153 and CEA in the first aspect.
Advantageous effects
Compared with the prior art, the test strip can detect CA125, CA153 and CEA tumor markers simultaneously, reduces the degree of difference of diagnosis results when detecting single tumor markers, improves the diagnosis efficiency and can meet the requirements of clinical diagnosis.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.
FIG. 1 is a schematic diagram of a test strip according to the present application;
Reference numerals: 1-sample pad, 2-latex pad, 3-chromatographic membrane, 301-labeling line, 302-detection line, 303-quality control line, 4-absorbent paper, and 5-bottom plate.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the exercise of inventive faculty, are intended to be within the scope of the invention.
The materials involved in the examples are all commercially available by conventional means.
Example 1
CA125, CA153 and CEA combined detection immunochromatography reagent strip
The kit comprises a sample pad 1, a latex pad 2, an NC film 3, absorbent paper 4 and a bottom plate 5, wherein the sample pad 1, the latex pad 2, the NC film 3 and the absorbent paper 4 are sequentially overlapped on the bottom plate 5, the latex pad 2 is coated with a mixture of a Eu microsphere marked CA125 monoclonal antibody I and a Bodipy fluoroborotic microsphere marked CEA monoclonal antibody I, a marking line 301 of the NC film 3 is coated with an Alexa Fluor 647 marked CA153 monoclonal antibody I, a detection line 302 of the NC film 3 is coated with a CA125 monoclonal antibody II, a CA153 monoclonal antibody II and a CEA monoclonal antibody II, and a quality control line 303 of the NC film 3 is coated with a goat anti-mouse IgG antibody.
In this example, the concentration of the Eu microsphere-labeled CA125 monoclonal antibody I, bodipy and the fluorine boron microsphere-labeled CEA monoclonal antibody I coated on the latex pad 2 is 0.5mg/mL. The concentration of Alexa Fluor labeled CA153 monoclonal antibody I coated on the label line 301 on NC membrane 3 was 0.5mg/mL. The concentration of CA125 monoclonal antibody II, CA153 monoclonal antibody II and CEA monoclonal antibody II coated on the detection line 302 of NC membrane 3 was 2.0mg/mL, 1.5mg/mL and 2.0mg/mL, respectively. The control line 303 on NC membrane 3 was coated with goat anti-mouse IgG antibody at a concentration of 1.0mg/mL.
In the embodiment, the CA125 monoclonal antibody I on the latex pad 2 is coupled with SA-Eu microsphere marks, and the CEA monoclonal antibody I is connected with SA-Bodipy fluorine boron microsphere marks; CA153 monoclonal antibody I on the labeled line 301 of NC membrane 3 was linked to Alexa Fluor 647.
The preparation process of the CA125, CA153 and CEA combined detection immunochromatography reagent strip comprises the following steps:
1.1 preparation of SA-Eu microsphere-labeled CA125 monoclonal antibody I
Adding 2.0mg of biotin-labeled CA125 monoclonal antibody I into 0.4mL of SA-Eu microsphere, incubating for 30 minutes at 37 ℃, centrifuging to obtain precipitate, washing the precipitate with a labeled washing buffer solution for 2 times, centrifuging for 2 times, removing unbound antibody-biotin from supernatant, finally taking the precipitate to be resuspended in 0.4mL of labeled preservation buffer solution A, finally preparing 2.0mg/mL of SA-Eu microsphere-labeled CA125 monoclonal antibody I, and preserving at 2-8 ℃ for later use.
1.1.1 Preparation of SA-Eu fluorescent microspheres
Eu microspheres were washed 2 times with 20mM MES buffer, centrifuged to remove the buffer, re-dissolved with 20mM MES buffer, the final concentration of Eu microspheres was set to 5mg/mL, 1-3-dimethylaminopropyl (EDC) 20uL at a concentration of 10mg/mL and N-hydroxysuccinimide (NHS) 40uL at a concentration of 10mg/mL were added to the re-dissolved microspheres, incubated in a shaker at 30℃for 30 minutes, the supernatant was removed by centrifugation, and the solubilized product was resuspended with 20mM MES buffer.
The labeling ratio of Streptavidin (SA) to Eu microspheres was 1mg: adding 5uL of SA solution with the final concentration of 2.5mg/mL, continuing to perform shaking table reaction for 2 hours at the temperature of 30 ℃ to obtain SA-Eu microspheres, washing the SA-Eu microspheres for 2 times by using a marked washing buffer solution, and adding 20mM MES buffer solution for resuspension, dissolution and preservation for later use.
1.1.2 Preparation of biotin-labeled CA125 monoclonal antibody I
CA125 monoclonal antibody I was combined with biotin labelling reagent at 1mg:3uL was mixed, incubated at 37℃for 30 minutes, and dialyzed against 100mMPB for 48 hours to obtain biotin-labeled CA125 monoclonal antibody I.
1.2 Preparation of SA-Bodipy fluoroboro microsphere-labeled CEA monoclonal antibody I
The preparation steps of the SA-Bodipy fluorine boron microsphere marked CEA monoclonal antibody I are the same as those of the step 1.1, except that Eu fluorescent substances are replaced by Bodipy fluorine boron microsphere fluorescent substances, and the concentration of the prepared SA-Bodipy fluorine boron microsphere marked CEA monoclonal antibody I is 2.0mg/mL.
1.3 Preparation of Alexa Fluor 647-labeled CA153 monoclonal antibody I
CA153 monoclonal antibody I was combined with Alexa Fluor 647 at 1:6 molar ratio, incubation at 37℃for 30min, dialysis against 100mM PB for 72 h, finally obtaining Alexa Fluor 647-labeled CA153 monoclonal antibody I, and storage in a refrigerator at 4℃for further use.
1.4 Treating latex pad
The latex pad 2 is made of glass fiber materials, SA-Eu microsphere marked CA125 monoclonal antibody I is diluted to 0.5mg/mL by using a marked preservation buffer solution A4 times, bodipy fluorine boron microsphere marked CEA monoclonal antibody I is diluted to 0.5mg/mL by using a marked preservation buffer solution A4 times, the latex pad 2 is sprayed on the sample pad 1 at 1.2uL/cm respectively, and after spraying, the latex pad 2 is dried for 4-5 hours under the environment of 18-26 ℃ and humidity of 20% -28%.
1.5, Sample pad for treatment
The sample pad 1 is made of glass fiber material, and the sample pad 1 is treated by using sample pad treatment liquid, wherein the sample pad treatment liquid comprises the following components: 20mM PBS, 1wt% sucrose, 1wt% PVP, 0.05v/v% Tween-20, 0.2mg/mLHBR-5.
The whole sample pad 1 was cut to a size of 1.5cm (width) by 30cm (length), each sample pad 1 was fully impregnated with 300mL of sample point treatment liquid, dried under 25% humidity for 4 hours, sealed with a desiccant, and stored for later use.
1.6, Treatment of NC film
The Alexa Fluor 647-labeled CA153 monoclonal antibody I was diluted to 0.5mg/mL with the label holding buffer B, and 0.8uL/cm was sprayed onto the labeled line 303 of NC membrane 3, and after the coating was completed, NC membrane 3 was dried at room temperature and humidity of less than 30% for 3 hours to obtain NC membrane 3 for immunochromatography detection, for use.
1.0Mg/mL of goat anti-mouse IgG antibody is sprayed on a quality control line 303 of an NC film 3 at 0.8uL/cm by using a three-dimensional plane spot film metal spraying instrument, 2.0mg/mL of CA125 monoclonal antibody II, 1.5mg/mLCA153 monoclonal antibody II and 2.0mg/mL of CEA monoclonal antibody II are sprayed on a detection line 302 of the NC film 3 at 0.7uL/cm respectively on a detection line 302, after the antibodies are coated, the NC film 3 is dried for 3 hours under the conditions of room temperature and humidity lower than 30%, and the NC film 3 used for immunochromatography detection is obtained for standby.
1.7, Assembly
The sample pad 1, the latex pad 2, the NC film 3 and the absorbent paper 4 which are processed are sequentially overlapped and stuck on the bottom plate 5, and finally the test paper plate is cut into a specific structure of the corresponding test paper strip as shown in figure 1, wherein the absorbent paper 4 and the bottom plate 5 are common components in the field.
1.8 Preparation of buffer
The buffer used in the above series of steps was prepared as follows:
1.8.1, 20mM MES buffer
Weighing MES 1.065g, dissolving with distilled water, adjusting pH to 6.5 with NaOH, autoclaving, and storing at 4deg.C.
1.8.2, Label wash buffer
1ML of Tween-20 was dissolved in 100mL of 20mM MES buffer, autoclaved and stored at 4℃for further use.
1.8.3, Marker preservation buffer A
Weighing 0.375g of glycine, 1g of bovine serum albumin, 1g of PVP and 10g of sucrose, dissolving in distilled water, regulating pH to 7.2-7.5 by NaOH, and finally keeping distilled water to 100mL, filtering in 0.22 um sterile condition, and preserving at 4 ℃ for later use.
1.8.4, Marker preservation buffer B
Weighing 1.125g of glycine, 3g of bovine serum albumin, 1g of PVP and 3g of sucrose, dissolving in distilled water, regulating pH to 7.2-7.5 by NaOH, finally fixing the volume of distilled water to 100mL, aseptically filtering with 0.22 um, and preserving at 4 ℃ for later use.
The invention also discloses a CA125, CA153 and CEA combined detection kit containing the test strip.
Example 2
The only difference from example 1 is that the antibody coated on the control line 303 on the test strip is a rabbit anti-mouse IgG antibody.
The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, but rather as providing for the use of additional embodiments and advantages of all such modifications, equivalents, improvements and similar to the present invention are intended to be included within the scope of the present invention as defined by the appended claims.
Claims (7)
1. The CA125, CA153, CEA combined detection immunochromatography test strip comprises a sample pad (1), a latex pad (2), an NC film (3), water absorbing paper (4) and a bottom plate (5), wherein the sample pad (1), the latex pad (2), the NC film (3) and the water absorbing paper (4) are sequentially overlapped on the bottom plate (5), and the immunochromatography test strip is characterized in that Eu microsphere marked CA125 monoclonal antibody I and Bodipy fluorine boron microsphere marked CEA monoclonal antibody I are coated on the latex pad (2); the marking line (301) of the NC film (3) is coated with Alexa Fluor 647 marked CA153 monoclonal antibody I; the detection line (302) of the NC film (3) is coated with a CA125 monoclonal antibody II, a CA153 monoclonal antibody II and a CEA monoclonal antibody II; the quality control line (303) of the NC film (3) is coated with a goat anti-mouse IgG antibody or a rabbit anti-mouse IgG antibody.
2. The immunochromatographic test strip according to claim 1, wherein the Eu-microsphere-labeled CA125 monoclonal antibody I is obtained by coupling reaction of a streptavidin Eu microsphere and a biotin-labeled CA125 monoclonal antibody I.
3. The immunochromatographic test strip according to claim 1, wherein the Bodipy fluoroboro microsphere marked CEA monoclonal antibody I is obtained by coupling reaction of streptavidin Bodipy fluoroboro microsphere and biotin marked CA125 monoclonal antibody I.
4. The immunochromatographic test strip according to claim 1, wherein the Alexa Fluor 647-labeled CA153 monoclonal antibody I is obtained by proportionally mixing Alexa Fluor 647 and CA153 monoclonal antibody I and dialyzing.
5. The immunochromatographic test strip according to claim 1, wherein the coating concentrations of the CA125 monoclonal antibody II, the CA153 monoclonal antibody II and the CEA monoclonal antibody II on the detection line (302) of the NC membrane (3) are respectively: 2.0mg/mL, 1.5mg/mL and 2.0mg/mL; the quality control line (303) of the NC film (3) is coated with 1.0mg/mL of goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody at 0.8 uL/cm.
6. A method for preparing an immunochromatographic test strip according to claim 1 to 5, which is characterized in that,
Comprising
Preparing a Eu microsphere marked CA125 monoclonal antibody I;
Preparing a Bodipy fluorine boron microsphere marked CEA monoclonal antibody I;
Preparing Alexa Fluor 647 marked CA153 monoclonal antibody I;
Coating a latex pad (2) with a Eu microsphere marked CA125 monoclonal antibody I and a Bodipy fluorine boron microsphere marked CEA monoclonal antibody I;
Coating an Alexa Fluor 647 marked CA153 monoclonal antibody I on a marked line (301) of an NC film (3), coating a CA125 monoclonal antibody II, a CA153 monoclonal antibody II and a CEA monoclonal antibody II on a detection line (302) on the NC film (3) in sequence, and coating a goat anti-mouse IgG antibody or a rabbit anti-mouse IgG antibody on a quality control line (303) of the NC film (3);
the sample pad (1), the latex pad (2), the NC film (3) and the water absorbing paper (4) prepared in addition are sequentially overlapped and fixed on the bottom plate (5).
7. A kit comprising the CA125, CA153, CEA combined detection immunochromatographic strip of any one of claims 1-5.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118275681A true CN118275681A (en) | 2024-07-02 |
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