CN117907608A

CN117907608A - Claudin2 protein detection method and related products and application thereof

Info

Publication number: CN117907608A
Application number: CN202410310425.6A
Authority: CN
Inventors: 陈菲; 霍如松; 李振军; 王莹
Original assignee: Hunan Herui Biotechnology Co ltd; Suzhou Herui Biotechnology Co ltd
Current assignee: Hunan Herui Biotechnology Co ltd; Suzhou Herui Biotechnology Co ltd
Priority date: 2024-03-19
Filing date: 2024-03-19
Publication date: 2024-04-19
Anticipated expiration: 2044-03-19
Also published as: CN117907608B

Abstract

The invention discloses a detection method of Claudin2 protein, and a related product and application thereof, and relates to the field of biology. The invention provides an extract, which comprises cathepsin S or elastase 1 and cathepsin E, and can be used for effectively extracting Claudin2 protein from a fecal sample, so that the Claudin2 protein is dissolved in a fecal supernatant, thereby realizing detection of target protein Claudin2 from the fecal sample and realizing noninvasive effective detection of Claudin2 protein.

Description

Claudin2 protein detection method and related products and application thereof

Technical Field

The invention relates to the field of biology, in particular to a detection method of Claudin2 protein, and a related product and application thereof.

Background

Human blocking protein 2 (Claudin 2, CLDN 2) is a four transmembrane protein comprising four transmembrane domains (TM 1-4), an intracellular N-and C-terminus and two extracellular loops (ECL 1 and ECL 2). ECL1 comprises four β -strands and one extracellular helix (ECH), ECL2 comprises one β -strand and one cell surface exposed partial transmembrane domain.

Claudin2 is expressed lowly in intestinal epithelium of healthy adults, and is significantly elevated in intestinal epithelium of patients with inflammatory bowel disease and celiac disease. Since it is a membrane structural protein, and is insoluble in fecal supernatant, currently, detection of Claudin2 is mainly immunohistochemical methods to perform immunohistochemical detection of sections of intestinal epithelium or detection of Claudin2 gene product in the intestinal contents. However, immunohistochemical detection is invasive, requires endoscopic biopsy of intestinal mucosal tissue, and has limited application range. For example, claudin2, disclosed in KR1020210140215, attorney docket 2021-10-20, can be detected by PCR in a colon lavage fluid from a laboratory mouse. The molecular detection method is strictly limited by the detection site because of the need for quantitative PCR instruments and the need for detection in a specialized PCR laboratory. The existing Claudin2 has the problems that the detection process is complicated, and the accurate quantification cannot be realized.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a detection method of Claudin2 protein, and a related product and application thereof.

The invention is realized in the following way:

in a first aspect, embodiments of the present invention provide a method for detecting or extracting a Claudin2 protein, comprising detecting Claudin2 or extracting Claudin2 from a fecal sample for non-disease diagnosis purposes using an extract; the active ingredients of the extract liquid comprise protease; the protease comprises any one of the following components: cathepsin S; elastase 1 and cathepsin E.

In a second aspect, embodiments of the present invention provide a reagent combination or kit comprising: the extract as described in the previous examples.

In a third aspect, embodiments of the present invention provide the use of an extract as described in the preceding embodiments for the preparation of a product for detecting Claudin2 or extracting Claudin2 or for aiding in the diagnosis of a disease associated with abnormal levels of Claudin 2.

The invention has the following beneficial effects:

according to the invention, through a special extract formula, claudin2 protein can be effectively extracted from a fecal sample, so that the Claudin2 protein is dissolved in a fecal supernatant, thereby realizing detection of target protein Claudin2 from the fecal sample and realizing noninvasive effective detection of Claudin2 protein.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the structure of the test strip in example 3.

Reference numerals: 1-sample pad; 2-a bonding pad; 3-nitrocellulose membrane; 4-water absorbing paper; 5-detection line (T line); 6-quality control line (C line).

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

In one aspect, embodiments of the present invention provide a method for detecting or extracting a Claudin2 protein, comprising detecting Claudin2 or extracting Claudin2 from a fecal sample for non-disease diagnosis purposes using an extract; the active ingredients of the extract liquid comprise protease; the protease comprises any one of the following components: cathepsin S; elastase 1 and cathepsin E.

The combination of elastase 1 and cathepsin E and cathepsin S have the same efficacy, and can simultaneously dissociate a target sequence from a Claudin2 protein sequence (which can be referred to as SEQ ID NO: 1) contained in a fecal sample and serve as a target for detecting the Claudin2 protein, thereby realizing qualitative or quantitative detection of the Claudin2 protein in the fecal sample. Compared with the prior art, the method only can realize the detection of the Claudin2 in the blood sample, and the extraction liquid and the detection method provided by the embodiment of the invention can realize the accurate detection of the Claudin2 protein with high efficiency and low cost under the conditions of no wound and no special equipment.

The target sequence comprises any one or more of polypeptide 2 and polypeptide 3. The amino acid sequence of the polypeptide 2 is shown as SEQ ID NO. 3, and the amino acid sequence of the polypeptide 3 is shown as SEQ ID NO. 4.

In some embodiments, the protease is cathepsin S. Cathepsin S (C01.034: CATHEPSIN S), which is capable of simultaneously cleaving the N-and C-termini of the ECL1 polypeptide in the Claudin2 protein sequence, and more importantly, there are no other cleavage sites for this enzyme within ECL1 (polypeptide 1). Specifically, the cathepsin S can be subjected to enzyme digestion between two Leus among Met Leu+Leu Pro at the 25 th-28 th sites of the N end of the ECL1 polypeptide sequence, and can be subjected to enzyme digestion between Gly and Leu among Leu-Gly+Leu-Pro at the 71 th-74 th sites of the C end. Here, "+" represents the cleavage site.

In some embodiments, where the protease comprises cathepsin S, the protease further comprises anthrax lethal factor (M34.001: anthrax lethal factor). Leu-Gly+Leu-Pro at positions 71-74 of the C-terminal of the ECL1 polypeptide sequence is also the cleavage site for anthrax lethal factor. The lethal factor (lethal factor, LF) is a metalloprotease capable of hydrolyzing mitogen-activated protein kinase (mitogen-ACTIVATED PROTEIN KINASE, MAPK) family, and is one of the important components of anthrax toxin (toxin). Anthrax LF consists of four domains: domain i is primarily responsible for binding to protective antigen (protective antigen, PA); domain ii is the primary functional region; domain iii is highly variable and capable of binding specifically to a substrate; domain iv contains a zinc ion binding sequence that exerts toxic effects on proteins.

The cathepsin S has the function of identifying the N end and the C end of the enzyme cutting target region, and the target sequence (polypeptide 2, the sequence of which is shown as SEQ ID NO: 3) can be dissociated from Claudin2 protein by using the cathepsin S alone as the protease. The anthrax lethal factor can identify the C end of a target area, does not cut other sites for identifying the target area, can be combined with cathepsin S to be used as protease, has no obvious difference with the effect of independently adopting the cathepsin S, and can also obtain the polypeptide 2 after enzyme cutting.

In some embodiments, the protease is a mixture of cathepsin S and anthrax lethal factor.

In other embodiments, the protease is a mixture of elastase 1 and cathepsin E. The elastase 1 (S01.153: elastase-1) can be used for enzyme digestion of Thr and Ser between 31-34 sites Lys-Thr+Ser-Ser at the N end of the ECL1 polypeptide sequence, and the cathepsin E (A01.010: CATHEPSIN E) can be used for enzyme digestion of Leu and Leu between 69-72 sites Thr-Leu+Leu-Gly at the C end, so that the target sequence polypeptide 3 is obtained, and the amino acid sequence of the polypeptide 3 is shown as SEQ ID NO 4.

With respect to the specific proteases defined in the examples of the present invention, the C-terminus of the ECL1 polypeptide may also be cleaved by other proteases, such as cathepsin B (C01.060: CATHEPSIN B), cathepsin L (C01.032: CATHEPSIN L) and cathepsin K (C01.036: CATHEPSIN K); however, the method is not suitable for the detection method provided by the invention, and the qualitative or quantitative detection of the Claudin2 protein cannot be effectively realized by applying the method to the invention.

In some embodiments, the final concentration of cathepsin S in the extract is 0.05-10 ng/mL. The final concentration may specifically be in the range of any one or between any two of 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10ng/mL.

In some embodiments, the final concentration of elastase 1 in the extract is 0.01-20 ng/mL. The final concentration may specifically be in the range between any one or any two of 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20ng/mL.

In some embodiments, the final concentration of cathepsin E in the extract is 0.01-10 ng/mL. The final concentration may specifically be in the range between any one or any two of 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10ng/mL.

In some embodiments, the final concentration of anthrax lethal factor in the extract is 0.01-5 ng/mL. The final concentration may specifically be in the range between any one or any two of 0.01, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5ng/mL.

In some embodiments, the extract further comprises: any one or more of a buffer, dithiothreitol, and a surfactant. The buffer solution, dithiothreitol and surfactant belong to conventional additive components in the extract, and the main inventive concept of the invention is to determine the active ingredient protease in the extract.

In some embodiments, the buffer is selected from the group consisting of: any one of Tris-HCl buffer, PBS buffer and borate buffer.

In some embodiments, when the buffer is Tris-HCl buffer, the final concentration of Tris-HCl in the extract may be 5-90 mM, and specifically may be in the range of between any one or any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 mM; the pH may be 5 to 8, and specifically may be any one or any two of 5, 5.5, 6, 6.5, 7, 7.5, and 8.

In some embodiments, when the buffer is a PBS buffer, the final concentration of PBS in the extract may be 1-50 mM, specifically may be in the range of between any one or any two of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM; the pH may be 5.5 to 7.5, and specifically may be in a range between any one or any two of 5.5, 6, 6.5, 7, and 7.5.

In some embodiments, when the buffer is a borate buffer, the final concentration of borate in the extract may be 2 to 100mM, and specifically may be in the range of between any one or any two of 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mM. The pH may be 6 to 9, and specifically may be any one or any two of 6, 6.5, 7, 7.5, 8, 8.5 and 9.

In some embodiments, the surfactant comprises: any one or more of tween 20, tween 80, triton X-100, triton X-405, tetronic1307, BRIIJ and SDS.

In some embodiments, the volume fractions (final concentrations) of tween 20, tween 80, triton X-100 and triton X-405 in the extract may independently be 0.01% -5%. Specifically, the ratio may be in a range between any one or any two of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%.

In some embodiments, the mass fraction of tetronic1307, BRIIJ, and SDS in the extract may independently be 0.01% -5%. Specifically, the ratio may be in a range between any one or any two of 0.01%, 0.02%, 0.04%, 0.06%, 0.08%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% and 5%.

In some embodiments, the final concentration of Dithiothreitol (DTT) in the extract is 0.5-20 mm. The final concentration may specifically be in the range between any one or any two of 0.5, 1,2, 3, 4,5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20mM.

In some embodiments, the step of detecting Claudin2 comprises: and mixing the extract with a sample to be detected, centrifuging the mixed product, and taking supernatant for detection.

In some embodiments, the step of detecting Claudin2 comprises: mixing the extract with a sample to be detected, filtering the mixture, and taking filtrate for detection.

In some embodiments, the method of detecting comprises: any one of enzyme-linked immunosorbent assay, immunochromatography, chemiluminescence and turbidimetry.

In some embodiments, the step of taking the supernatant or filtrate for detection further comprises: capturing the target object by using a capture antibody and/or detecting the signal of the target object by using a detection antibody;

Wherein the antigen of the capture antibody and/or the detection antibody is independently an antigen polypeptide selected from any one of polypeptide 1, polypeptide 2 and polypeptide 3; the amino acid sequences of the polypeptide 1, the polypeptide 2 and the polypeptide 3 are shown as SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4 in sequence.

The polypeptides 1-3 are three sections of amino acid sequences with higher immunogenicity, and Claudin2 antigen protein can be obtained through gene recombination, transfection and expression and can be used as a standard in a detection kit. Meanwhile, any one or more of the polypeptides 1-3 are immunized, antibodies with high specificity and Claudin2 protein resistance can be obtained through screening, and the antibodies can be monoclonal antibodies, polyclonal antibodies or antigen binding fragments. The antibody obtained by screening is combined with a solid phase carrier and can be used as a capturing antibody for capturing a target object in the supernatant of the excrement, and/or the antibody obtained by screening is combined with a marker and can be used as a detection antibody for detecting the target object.

In another aspect, embodiments of the present invention also provide a reagent combination or kit comprising the extract as described in any of the previous embodiments.

The term "plurality" herein refers to two or more.

In another aspect, the present invention provides a method for detecting Claudin2 or extracting Claudin2, or for aiding in diagnosing a disease associated with abnormal levels of Claudin2, comprising applying the extract of any of the above embodiments to the preparation of a product for detecting Claudin2 or extracting Claudin 2.

In some embodiments, the disorder associated with abnormal levels of Claudin2 comprises: any one or more of inflammatory bowel disease or celiac disease. The Claudin2 level comprises the Claudin2 protein level and/or mRNA level.

In some embodiments, the product comprises: a reagent or kit.

On the other hand, the embodiment of the invention also provides the application of the extract liquid in any embodiment in constructing a prediction model for auxiliary prediction or auxiliary diagnosis of inflammatory bowel disease or celiac disease.

In another aspect, the embodiment of the invention further provides an antigen polypeptide, wherein the antigen polypeptide is selected from any one of polypeptide 1, polypeptide 2 and polypeptide 3; the amino acid sequences of the polypeptide 1, the polypeptide 2 and the polypeptide 3 are shown as SEQ ID NO.2, SEQ ID NO. 3 and SEQ ID NO. 4 in sequence.

In another aspect, embodiments of the present invention also provide a polypeptide composition comprising any two or three of polypeptide 1, polypeptide 2, and polypeptide 3. The amino acid sequences of the polypeptide 1, the polypeptide 2 and the polypeptide 3 are shown as SEQ ID NO. 2, SEQ ID NO.3 and SEQ ID NO.4 in sequence.

In another aspect, the antigenic polypeptide or polypeptide composition of the preceding examples is used for the detection of Claudin2 or for the extraction of Claudin2 from fecal samples for non-disease diagnostic purposes.

In another aspect, the use of the antigenic polypeptide or polypeptide composition of the preceding examples in the manufacture of a product for detecting Claudin2 or extracting Claudin2 or for aiding in the diagnosis of a disease associated with abnormal levels of Claudin 2.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Samples taken in the subsequent examples: inflammatory Bowel Disease (IBD) group and healthy control group stool samples were 50 each. Specifically, the inflammatory bowel disease group is a definitive sample sent to the detection center, of which 41 cases of crohn's disease and 9 cases of ulcerative colitis; age 10-42 years old; 29 men and 21 women. The healthy control group is fecal sample of staff and family members of the center, and the age is 9-48 years; 30 men and 20 women.

Example 1

This example provides a plurality of sets of extracts for extracting Claudin2 protein from fecal samples, as follows.

TABLE 1 composition of extract and final concentration of the composition

Remarks: the concentrations of the components in the table are their final concentrations in the extract.

Example 2

The embodiment provides a kit and a detection method for detecting Clandin < 2 > based on an enzyme-linked immunosorbent assay (ELISA).

1. Kit for detecting a substance in a sample

The kit comprises: ELISA plate, calibrator, quality control, enzyme conjugate, substrate, cleaning solution, stop solution and fecal extract (extracts 1-6 in example 1).

Wherein the enzyme conjugate is horseradish peroxidase (HRP) labeled with anti-polypeptide 2 (SEQ ID NO: 3) murine monoclonal antibody. The ELISA plate is coated with goat polyclonal antibody against ECL1 protein. The calibrator was ECL1 protein at 6 concentration gradients.

The preparation method of partial reagent in the kit comprises the following steps:

(1) And (3) coating an ELISA plate: goat polyclonal antibody against ECL1 protein was diluted to 5 μg/mL with CB buffer, 100 μl per well coated with microplate, overnight at 4 ℃. The liquid in the wells was dried, incubated with 250. Mu.L of blocking buffer containing BSA per well for 2 hours at 37℃and the blocking solution was removed by pipetting and oven dried at 37℃for further use.

(2) Anti-polypeptide 2 murine mab labeled horseradish peroxidase (HRP): 2mg of HRP is weighed and placed in a centrifuge tube, 1mL of pure water is added for dissolution, 1mL of NaIO ₄ solution with the concentration of 10mg/mL is added, and the reaction is carried out for 20min at 4 ℃ in a dark place. 0.05mL of ethylene glycol was added and the reaction was carried out at room temperature for 10min. And (5) ultrafiltration and purification. To this was added 2mg of anti-polypeptide 2 murine monoclonal antibody, which was reacted at 4℃for 3 hours in the absence of light, 0.2mL of NaBH ₄ solution at a concentration of 5mg/mL was added, and the mixture was homogenized and dialyzed overnight against 0.05M CB. And taking out the labeled enzyme-labeled antibody, adding the equal volume of glycerol, and storing at the temperature of minus 20 ℃ for standby.

2. Detection method

(1) 5ML of the extract was added to 10mg of feces, the mixture was thoroughly mixed, centrifuged at 3000rpm for 5min, and the supernatant was collected for detection. The ELISA plate was added at 100. Mu.L per well, incubated at 37℃for 2 hours, and the plate was washed 3 times.

(2) Enzyme conjugate was added and incubated for 1 hour and the plates were washed 4 times.

(3) Adding a substrate TMB into the ELISA plate, incubating for 15 minutes, adding a stop solution to stop the chromogenic reaction, detecting an OD value by using a wavelength of 450nm, comparing the OD value with an OD value of a standard substance, and calculating the concentration of Claudin2 (ECL 1) in a sample to be detected.

3. Detection effect verification of kit

Experimental method

The kit and the detection method provided in this example were used as an experimental group, and a control group was set at the same time, and 100 samples of stool samples were detected (50 samples of stool samples of inflammatory bowel disease IBD group and healthy control group).

In addition, a group of control is provided, the detection method of the control group is a method for detecting Claudin2 RNA in a sample based on PCR, and the fecal nucleic acid extraction reagent is purchased from a root organism.

Experimental results

Table 2 experimental results

The ELISA reagent feces provided by the embodiment of the invention examine the extract liquid 1-6. As shown in Table 1, the results of the extraction solutions 1 to 3 all achieved good results, the positive coincidence rates were 76%, 78% and 76%, the negative coincidence rates were 96%, 92% and 90%, and the total coincidence rates were 86%, 85% and 83%, respectively. The positive, negative and total compliance rates of the control PCR method were 68%, 58% and 63%, respectively. The results show that cathepsin S alone or in combination with anthrax lethal factor, and cathepsin E in combination with elastase 1, are superior to control PCR detection methods. Wherein the formula of the extract 1 is the simplest, and has the advantage of high cost performance.

The detection results of the extraction solutions 4-6 are inferior to that of the PCR method.

Example 3

The embodiment provides a kit and a detection method for quantitatively detecting Claudin2 based on fluorescence chromatography.

1. Kit for detecting a substance in a sample

The kit comprises: fluorescent chromatography quantitative detection card, calibration ID card and fecal extract (extract 1, 7-13 in example 1).

The fluorescent chromatography quantitative detection card is an aluminum foil bag sealed package and comprises a detection card and a drying agent. The detection card comprises a plastic card shell test strip. The test paper has a structure shown in figure 1, and is characterized in that a nitrocellulose membrane 3, a sample pad 1, a bonding pad 2 and absorbent paper 4 are stuck on a PVC bottom plate, the sample pad 1 is laminated at one end of the bonding pad 2, and the other end of the bonding pad 2 and the absorbent paper 4 are respectively laminated at two ends of the nitrocellulose membrane 3; the binding pad 2 is provided with a time-resolved fluorescent microsphere marked anti-ECL 1 protein chicken polyclonal antibody (chicken IgY polyclonal antibody); the nitrocellulose membrane 3 is provided with a detection line 5 (T line) and a quality control line 6 (C line), the detection line 5 (T line) is coated with anti-polypeptide 3 (SEQ ID NO: 4) rabbit monoclonal antibody, and the quality control line 6 (C line) is coated with goat anti-chicken IgY polyclonal antibody (Goat anti-CHICKEN IGY polyclonal Antibody).

The preparation method of the kit comprises the following steps:

(1) Time-resolved fluorescent microspheres label anti-ECL 1 protein chicken polyclonal antibodies.

200. Mu.L (0.2 μm, 1%) of time-resolved fluorescent microspheres were centrifuged at 13500rpm at 4℃for 15min and the supernatant was discarded. Adding 10mg/ml EDC, reacting for 15min, centrifuging, discarding supernatant, adding 400 μl of borate buffer (20 mM, pH 8.0), and mixing with ultrasound for 15 s; adding 80 mug of anti-ECL 1 protein chicken polyclonal antibody, and reacting for 2 hours at 25 ℃; centrifuging, discarding the supernatant, adding a sealing solution, and reacting for 1h; centrifuging, discarding supernatant, adding 400 μl of borate buffer solution, mixing well by ultrasound, labeling, and preserving at 4deg.C.

(2) Preparation of conjugate pad and sample pad

The polyester film was soaked in a buffer solution (formula: 20mM pH 8.0 PBS containing 5% by mass of BSA, 0.5% by mass of S9 and 3% by mass of trehalose) containing a surfactant for pretreatment, and dried at 37℃overnight to prepare a conjugate pad; taking the prepared bonding pad, spraying time-resolved fluorescent microsphere marked with anti-ECL 1 protein chicken polyclonal antibody onto the pretreated bonding pad by using a gold mark HM3035 metal spraying film-dividing instrument, drying at 37 ℃ for 4 hours, and cutting into 30 multiplied by 0.8cm after drying to prepare the sample pad. The specific binding pad is prepared.

The glass cellulose membrane was immersed in a buffer solution (formula: 50mM pH 8.0 BB containing 2% by mass of BSA and 0.5% by mass of S9) containing a surfactant, and then, the membrane was pre-sealed, dried overnight at 37℃and cut into a sample pad of 30X 1.6 cm.

(3) Coating T-line and C-line on nitrocellulose film (NC film)

NC film is pasted on the appointed position of the back lining bottom plate, anti-polypeptide 3 (SEQ ID NO: 4) rabbit monoclonal antibody and sheep anti-chicken IgY polyclonal antibody (Goat anti-CHICKEN IGY polyclonal Antibody) are respectively diluted to 1mg/mL and 0.5mg/mL, and then the mixture is uniformly sprayed on the NC film by a gold mark HM3035 metal spraying film-dividing instrument to prepare T line and C line, and the mixture is dried at 37 ℃ overnight.

(4) Assembly

Laminating the bonding pad obtained in the step (2) on one end of the nitrocellulose membrane obtained in the step (3), fixedly laminating a water absorbing membrane on the other end of the nitrocellulose membrane, laminating the sample pad obtained in the step (2) on the other end of the bonding pad, cutting by a membrane cutting instrument according to the width of 4mm, and loading into a chromatographic strip shell to obtain a finished product (a detection card).

(5) Making a calibration curve

And (3) adding 1 set of calibrator 1 into a detection card, reacting for 10min, detecting by using a fluorescence reader FIC-S100 to obtain a calibration curve, and preparing the calibration ID card by HMreader software.

2. Detection method

After the excrement sample is sampled by the excrement sampling rod of the excrement processing pipe (5 mL of extraction liquid is added into 10mg of excrement), the excrement sample is evenly mixed and dissolved by shaking, and the extracted sample extraction liquid is extruded into a test tube. And adding 80 mu L of the sample into the sample adding hole, reacting for 10min, and then placing the sample into a fluorescence reader for detection to obtain the content of Claudin2 (ECL 1) in the sample.

3. Detection effect verification of kit

Experimental method

The detection method and detection reagent of the control group were the same as in example 2.

Experimental results

TABLE 3 experimental results

As can be seen from the results in Table 3, the results of extracts 1, 8 and 11 were consistent, the protease content was consistent among the three groups, and the changes in factors such as buffer system, surfactant and DTT content were not significantly different from the results. The chromatographic results of the 8 extracts were all better than the control PCR method, with extracts 1, 8 and 11 being optimal.

Example 4

The embodiment provides a kit for detecting Clandin < 2 > based on a magnetic particle chemiluminescence method and a detection method thereof.

1. Kit for detecting a substance in a sample

The kit comprises: fecal extract (extract in example 1), biotinylated anti-polypeptide 3 (SEQ ID NO: 4) murine mab, standard, acridinium ester-labeled anti-polypeptide 2 (SEQ ID NO: 3) rabbit mab, streptavidin magnetic particles, excitation solution and wash solution. Wherein, the calibrator is ECL1 protein with different concentration gradients.

(1) Preparation of biotinylated anti-polypeptide 3 murine monoclonal antibody

2Mg of anti-polypeptide 3 murine mAb was placed in a centrifuge tube, 80. Mu.L of activated biotin was added thereto, the reaction was carried out at 25℃for 30 minutes, and the reaction solution was taken out and dialyzed against 0.02M PBS overnight. Taking out the biotinylated anti-polypeptide 3 mouse monoclonal antibody, adding 0.05% Proclin300, and keeping at 2-8deg.C.

(2) Acridinium ester marked anti-polypeptide 2 rabbit monoclonal antibody

2Mg of anti-polypeptide 2 rabbit monoclonal antibody is taken according to the following antibody: acridine ester = 1:20 molar ratio the activated acridine ester was added and reacted for 30min at room temperature. The reaction solution was dialyzed and taken out, and 0.05% Proclin300 was added thereto at 2-8deg.C for use.

2. Detection method

(1) The fecal sample was mixed well with the extract of example 1 by shaking for 1min (5 mL extract was added per 10mg fecal sample), and the mixed product was filtered and the filtrate was used for detection.

(2) The model of the luminometer is as follows: kestesmart 500s. Setting detection parameters of a luminometer: and (3) adding 20 mu L of biotinylated anti-polypeptide 3 mouse monoclonal antibody 100 mu L and acridinium ester labeled anti-polypeptide 2 rabbit monoclonal antibody 100 mu L, incubating for 30min, washing for 4 times, and detecting.

(3) And (3) performing on-machine detection on the fecal sample extract and the calibrator, and calculating the concentration of Claudin2 (ECL 1) in the sample according to the luminescence value of the calibrator.

3. Detection effect verification of kit

Experimental method

Experimental results

Table 4 Experimental results

From the results, all formulations examined were superior to the control PCR method. There was no significant difference between the 8 formulations, with extracts 1, 15, 16 being particularly excellent.

Example 5

The embodiment provides a kit for detecting Clandin2 by a latex-enhanced immunoturbidimetry and a detection method thereof.

1. Kit for detecting a substance in a sample

The kit comprises: r1 reagent, R2 reagent and fecal extract (extract in example 1). Wherein the R1 reagent is MOPS buffer solution. The R2 reagent is a mixed solution of latex microspheres marked with anti-ECL 1 protein chicken polyclonal antibody and latex microspheres marked with anti-ECL 1 rabbit monoclonal antibody; the particle size of the latex microsphere marked with the anti-ECL 1 protein chicken polyclonal antibody is 300nm, and the particle size of the latex microsphere marked with the anti-ECL 1 rabbit monoclonal antibody is 100nm.

(1) Preparation of R1 reagent

The R1 reagent is MOPS buffer containing PEG6000 with the mass fraction of 1%, BSA with the mass fraction of 1% and Proclin300 with the volume fraction of 0.05%. Filtering with 0.45 μm membrane after the preparation, and packaging at 2-8deg.C.

(2) Preparation of R2 reagent

Preparation of latex microspheres of anti-ECL 1 protein chicken polyclonal antibody:

100. Mu.L (300 nm) of latex microspheres were taken and centrifuged at 13500rpm for 10min at 4℃and the supernatant was discarded. 0.04ml of NHS solution and 0.02ml of EDC solution are added, and the mixture is stirred and stirred evenly and reacted for half an hour at room temperature. Centrifuging and discarding the supernatant. 1mg of anti-ECL 1 protein chicken polyclonal antibody is added, and the mixture is stirred and mixed uniformly, and the mixture is subjected to rotary reaction at room temperature for 2 hours. Centrifuging and discarding the supernatant. Adding 1ml of sealing solution, mixing by ultrasonic vortex, performing rotary reaction at room temperature for 1 hour, centrifuging, discarding supernatant, adding microsphere preservation solution, mixing by ultrasonic, and standing at 2-8deg.C.

Preparation of latex microspheres of anti-ECL 1 rabbit monoclonal antibody:

100. Mu.L (100 nm) of latex microspheres were taken and centrifuged at 13500rpm for 20min at 4℃and the supernatant was discarded. 0.04ml of NHS solution and 0.02ml of EDC solution are added, and the mixture is stirred and stirred evenly and reacted for half an hour at room temperature. Centrifuging and discarding the supernatant. 1mg of anti-ECL 1 rabbit monoclonal antibody is added, vortex mixed evenly and rotate at room temperature for 2 hours. Centrifuging and discarding the supernatant. Adding 1ml of sealing solution, mixing by ultrasonic vortex, performing rotary reaction at room temperature for 1 hour, centrifuging, discarding supernatant, adding microsphere preservation solution, mixing by ultrasonic, and standing at 2-8deg.C.

Mixing the two latex microspheres according to the volume ratio of 1:1, and diluting the mixture by 10 times to prepare the R2 reagent.

2. Detection method

(1) After the fecal sample was mixed with the sample extract of example 1 by shaking for 1min, 5mL of the extract was added to each 10mg of feces, and the resulting feces extract was filtered for subsequent detection.

(2) The biochemical analyzer is a Michaelis BS-240 full-automatic biochemical analyzer. Setting detection parameters of a biochemical analyzer: the sample was added in an amount of 10. Mu.L, R1 was 120. Mu.L, R2 was 20. Mu.L, incubation time was 8, and reaction time (5, 16).

(3) And (3) performing machine detection on the fecal sample extract and the calibrator (ECL 1 proteins with 6 concentration gradients), and calculating the concentration of Claudin2 (ECL 1) in the sample according to the reactivity value of the calibrator.

3. Detection effect verification of kit

Experimental method

Experimental results

TABLE 5 experimental results

The positive compliance rate of the 4 extract liquid formulas is 68-72%, and has no obvious difference with the control PCR method, but the negative compliance rate is 86-92%, and the total compliance rate is 79-82%, both of which are obviously superior to the control method. The extract 1 has relatively lower positive coincidence rate and total coincidence rate than ELISA, fluorescence chromatography and magnetic particle luminescence in the method, and is considered to be slightly weaker in detection sensitivity than the prior three methods by turbidimetry. The extracts 18-20 are all superior to the control PCR method.

The sequences to which the invention relates are shown in the following table.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting or extracting Claudin2 protein is characterized by comprising the steps of detecting Claudin2 or extracting Claudin2 from a fecal sample by adopting an extraction solution for the purpose of non-disease diagnosis; the active ingredients of the extract liquid comprise protease; the protease comprises any one of the following components: cathepsin S; elastase 1 and cathepsin E.

2. The method according to claim 1, wherein the final concentration of cathepsin S in the extract is 0.05-10 ng/mL;

In the extract, the final concentration of the elastase 1 is 0.01-20 ng/mL;

in the extract, the final concentration of the cathepsin E is 0.01-10 ng/mL.

3. The method of claim 1, wherein when the protease comprises cathepsin S, the protease further comprises anthrax lethal factor;

in the extract, the final concentration of the anthrax lethal factor is 0.01-5 ng/mL.

4. A detection or extraction method according to any one of claims 1 to 3, wherein the extract further comprises: any one or more of a buffer, dithiothreitol, and a surfactant.

5. The method of detection or extraction of claim 4, wherein the buffer is selected from the group consisting of: any one of Tris-HCl buffer, PBS buffer and borate buffer;

the surfactant comprises: any one or more of tween 20, tween 80, triton X-100, triton X-405, tetronic1307, BRIIJ and SDS.

6. A method of detecting or extracting according to any one of claims 1 to 3, wherein the step of detecting Claudin2 comprises: mixing the extract with a sample to be detected, centrifuging or filtering the mixed product, and taking supernatant or filtrate for detection.

7. The method of claim 6, wherein the step of taking supernatant or filtrate for detection further comprises: capturing the target object by using a capture antibody and/or detecting the signal of the target object by using a detection antibody;

8. A method of detection or extraction according to any one of claims 1 to 3, wherein the method of detection comprises: any one of enzyme-linked immunosorbent assay, immunochromatography, chemiluminescence and turbidimetry.

9. A reagent combination or kit, comprising: the extract according to any one of claims 1 to 8.

10. Use of an extract as claimed in any one of claims 1 to 8 for the preparation of a product for detecting Claudin2 or extracting Claudin2 or for aiding in the diagnosis of diseases associated with abnormal levels of Claudin 2.