Detection kit for detecting calprotectin in human excrement

Abstract

The invention discloses a detection kit for detecting calprotectin in human excrement, the detection kit is composed of a sample diluent and detection test paper, the detection test paper is composed of a bottom plate, a nitrocellulose membrane, a conjugate pad, a sample pad and absorbent paper, the absorbent paper, the nitrocellulose membrane, the conjugate pad and the sample pad are sequentially overlapped on the bottom plate, a shell is arranged outside the detection test paper, and the shell is connected with the sample pad. An observation window and a sample adding hole are formed in the shell, and the position of the observation window is matched with the position of the nitrocellulose membrane; the position of the sample adding hole is matched with the position of the sample pad; the nitrocellulose membrane is sequentially provided with a T1 detection line coated with an anti-calprotectin monoclonal antibody, a T2 detection line coated with an anti-calprotectin monoclonal antibody and a quality control line C coated with a goat anti-chicken IgY antibody and / or a goat anti-mouse IgG antibody; a calprotectin monoclonal antibody marked by colloidal gold is solidified on the combination pad, and the sample diluent is prepared from sodium chloride, EDTA (Ethylene Diamine Tetraacetic Acid), PEG (Polyethylene Glycol) 20000, Proclin-300 and purified water.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics, and more specifically, this invention relates to a detection kit for detecting calprotectin in human feces. Background Technology

[0002] Calprotectin is a newly discovered marker of acute inflammation. First isolated from neutrophils in 1980, calprotectin is a heterodimer S100A8 / A9 protein complex formed in a calcium-dependent manner by S100A8 (MRP-8) and S100A9 (MRP-14) proteins. Calprotectin possesses anti-protease activity and exhibits heat resistance due to its ability to chelate zinc ions.

[0003] Calprotectin is a marker of neutrophil turnover and possesses various biological functions, including antimicrobial activity, immune regulation, antiproliferation, and signal transduction. Its levels rise in many inflammatory conditions. Calprotectin can be detected in plasma, urine, feces, cerebrospinal fluid, saliva, synovial fluid, and colon biopsy tissue. Studies have found that calprotectin levels in the feces of patients with inflammatory bowel disease (IBD) are significantly higher than those in patients with irritable bowel syndrome (IBS), and are positively correlated with the severity of the disease. Therefore, fecal calprotectin can serve as a diagnostic indicator to differentiate between IBD, colon cancer, and IBS patients. It also has important guiding significance for disease recurrence and treatment efficacy.

[0004] Studies by foreign scholars on the correlation between inflammatory bowel disease (IBD) and fecal calprotectin levels have shown that the average fecal calprotectin level is 175 (44–938) ug / g in patients with Crohn's disease (CD), 465 (61–1128) ug / g in patients with ulcerative colitis (UC), and 54 (16–1128) ug / g in patients with other bowel diseases, including irritable bowel syndrome (IBS). Fecal calprotectin levels are significantly correlated with endoscopic activity levels in IBD, and this correlation has been confirmed to be significant during both active and remission phases of the disease.

[0005] Currently, fecal calprotectin is the best indicator of intestinal mucosal inflammation in patients with inflammatory bowel disease (IBD). If endoscopy is based on fecal calprotectin levels, the rate of endoscopic examination in suspected IBD patients will decrease to 32%, reducing unnecessary colonoscopies.

[0006] Domestic scholars have also conducted numerous clinical studies, confirming that fecal calprotectin levels in patients with active ulcerative colitis (UC) are significantly higher than those in patients with colonic polyps and normal individuals. The level of fecal calprotectin can reflect the lesions of the intestinal mucosa and the severity of the patient's disease. Research by Li Xiaoguang et al. showed that, using a fecal calprotectin concentration greater than 50 ug / g as the cutoff value, the positive rate was 85% in the colorectal cancer group and 91.7% in the chronic inflammation group. Research by Ji Chunyan et al. showed that the median fecal calprotectin level, from highest to lowest, was as follows: infectious diarrhea group (median 515.21 ug / g), colorectal cancer group (median 162.47 ug / g), D-IBS group (median 35.02 ug / g), and normal control group (median 25.18 ug / g). This suggests that a fecal calprotectin level ≥70 ug / g should strongly suggest the possibility of infectious diarrhea or colonic tumors. Combined with the patient's clinical history, a preliminary diagnosis of infectious diarrhea or colonic tumors can be made.

[0007] Currently, the main methods for detecting calprotectin levels include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (chemiluminescence immunoassay), and colloidal gold immunochromatography (CIGC). While ELISA and chemiluminescence immunoassay can accurately measure calprotectin levels in feces, they are cumbersome, time-consuming, and require specialized equipment. Colloidal gold immunochromatography is simple, fast, and does not require specialized equipment. However, most colloidal gold products on the market for detecting calprotectin have sensitivity settings that do not meet clinical testing needs. Their claimed high sensitivity can lead to false positives, even in healthy samples. Furthermore, most products only differentiate between inflammatory bowel disease and healthy individuals or irritable bowel syndrome, offering no guidance on relapse risk after treatment or transplant rejection. Summary of the Invention

[0008] Objective of the Invention: To address the shortcomings of the prior art, this invention discloses a detection kit for calprotectin in human feces. The detection sensitivity setting of the test strip of this invention can reduce the probability of false positives, and by setting two detection lines, it can differentiate between inflammatory bowel disease and healthy individuals or irritable bowel syndrome. It can also provide clinical guidance for monitoring the risk of relapse after treatment and transplant rejection.

[0009] The sample diluent of this invention contains a variety of chemical components, which can improve the chromatography speed, eliminate the adverse effects of the detection reaction, reduce the false positive rate, and improve the detection accuracy.

[0010] Technical Solution: A detection kit for calprotectin in human feces, comprising a sample diluent and a test strip. The test strip consists of a base plate, a nitrocellulose membrane, a conjugate pad, a sample pad, and absorbent paper. The absorbent paper, the nitrocellulose membrane, the conjugate pad, and the sample pad are sequentially overlapped on the base plate. The test strip also has an outer shell with an observation window and a sample application hole. The position of the observation window is adapted to the position of the nitrocellulose membrane, and the position of the sample application hole is adapted to the position of the sample pad.

[0011] The nitrocellulose membrane is sequentially provided with a T1 detection line coated with anti-calprotectin monoclonal antibody, a T2 detection line coated with anti-calprotectin monoclonal antibody, and a quality control line C coated with goat anti-chicken IgY antibody and / or goat anti-mouse IgG antibody.

[0012] The binding pad is immobilized with a colloidal gold-labeled calprotectin monoclonal antibody.

[0013] The sample diluent consists of the following components:

[0014]

[0015]

[0016] The remainder is purified water, and the pH of the sample dilution is 7.0–7.6.

[0017] Furthermore, the detection threshold of the T1 detection line is 50 ug / g.

[0018] Furthermore, the detection threshold of the T2 detection line is 200 ug / g.

[0019] Furthermore, the interval between the T2 detection line and the T1 detection line is 4-6 mm, and the interval between the T2 detection line and the quality control line C is 4-6 mm.

[0020] Furthermore, the T1 detection line is coated with one or more of mouse anti-calprotectin monoclonal antibodies, goat anti-calprotectin monoclonal antibodies, and rabbit anti-calprotectin monoclonal antibodies. The coating concentration of the T1 detection line is 1.0–2.5 mg / ml, and the coating buffer is 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0021] Furthermore, the T2 detection line is coated with one or more of mouse anti-calprotectin monoclonal antibodies, goat anti-calprotectin monoclonal antibodies, and rabbit anti-calprotectin monoclonal antibodies. The coating concentration of the T2 detection line is 0.5–1.5 mg / ml, and the coating buffer is 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0022] Furthermore, the coating concentration of control line C was 1.0–2.0 mg / ml, and the coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0023] Furthermore, one end of the absorbent paper is pressed against one end of the nitrocellulose membrane at a distance of 1–2 mm, and / or

[0024] The absorbent paper is made of either glass fiber film or polyester film, preferably polyester film.

[0025] Furthermore, one end of the binding pad is pressed against the other end of the nitrocellulose membrane at a distance of 1–2 mm, and / or

[0026] The bonding pad is made of one of the following materials: glass fiber membrane, polyester membrane, or non-woven fabric, with glass fiber membrane being preferred.

[0027] Furthermore, the pad is immobilized with one or more of the following: mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody, and rabbit anti-calprotectin monoclonal antibody, and / or

[0028] The concentration of colloidal gold-labeled calprotectin monoclonal antibody is 10 μg / ml to 30 μg / ml, and / or

[0029] The particle size of colloidal gold is 20–50 nm.

[0030] Furthermore, the preparation process of the bonding pad is as follows:

[0031] 1) Monoclonal antibodies labeled with calprotectin:

[0032] Adjust the pH of the colloidal gold to 8.0–8.5 using 0.2M K₂CO₃ solution. Add calprotectin monoclonal antibody at a labeling concentration of 10–30 μg / ml. Stir and mix thoroughly for 30–60 min. Block with 20% BSA solution to a final blocking concentration of 0.5%–1%. Centrifuge and discard the supernatant. Reconstitute the precipitate with a reconstitution solution to half its original volume.

[0033] The reconstitution solution is a 0.02M Tris-HCl buffer solution containing 0.1-0.5% PEG20000, 0.5-1% casein, 1%-5% sucrose, and 0.05%-0.2% Tween 80, with a pH of 8.0-8.5.

[0034] 2) Using a gold sprayer, uniformly spray the gold-labeled calprotectin monoclonal antibody obtained in step 1) onto a glass fiber membrane, polyester membrane, or non-woven fabric. The gold spraying amount is 4ul / cm to 8ul / cm. Then, place it in a drying room at 37°C for 24 to 32 hours to dry.

[0035] Furthermore, the sample pad is pretreated with a sample pad treatment solution, which consists of the following components:

[0036]

[0037] The remainder is purified water, and the pH value of the sample pad treatment solution is 8.0 to 8.2.

[0038] Beneficial effects: The detection kit for detecting calprotectin in human feces disclosed in this invention has the following beneficial effects:

[0039] 1. It is easy to operate and does not require sophisticated instruments;

[0040] 2. The detection sensitivity setting of the test strip can reduce the probability of false positives, and by setting two detection lines, it is possible to distinguish between inflammatory bowel disease and healthy individuals or irritable bowel syndrome. At the same time, it can also provide clinical guidance for monitoring the risk of recurrence after treatment and transplant rejection. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the test strip.

[0042] Figure 2 This is a diagram illustrating a negative test result.

[0043] Figure 3 and Figure 4 This is a diagram illustrating a positive test result.

[0044] Figure 5 This is a diagram illustrating invalid test results.

[0045] Among them: 1-base plate

[0046] 2-Nitrocellulose membrane

[0047] 3-Absorbent Paper

[0048] 4-Conjunction Pad

[0049] 5-Sample Pad

[0050] 21-T1 testing line

[0051] 22-T2 testing line

[0052] 23-Quality Control Line C Detailed Implementation

[0053] The specific embodiments of the present invention are described in detail below.

[0054] The "range" disclosed in this invention is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10–50 is listed for a specific parameter, it is also expected that ranges of 10–40 and 20–50 are also included. Furthermore, if the minimum range values ​​are 1 and 2, and the maximum range values ​​are 3, 4, and 5, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0–5" means that all real numbers between "0–5" have been listed herein; "0–5" is merely a shortened representation of these numerical combinations.

[0055] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0056] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0057] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0058] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0059] Unless otherwise specified, the reaction will proceed under normal temperature and pressure conditions.

[0060] Unless otherwise specified, all parts or percentages are by weight or by weight percentage.

[0061] In this invention, all the substances used are known substances that can be purchased or synthesized by known methods.

[0062] In this invention, all the devices or equipment used are conventional devices or equipment known in the art and are readily available.

[0063] A test kit for detecting calprotectin in human feces comprises a sample diluent and a test strip. The test strip consists of a base plate, a nitrocellulose membrane, a conjugate pad, a sample pad, and absorbent paper. The absorbent paper, the nitrocellulose membrane, the conjugate pad, and the sample pad are sequentially overlapped on the base plate. The test strip also has an outer shell with an observation window and a sample application port. The position of the observation window corresponds to the positions of the T1 detection line, T2 detection line, and control line C on the nitrocellulose membrane. The position of the sample application port corresponds to the position of the sample pad.

[0064] The nitrocellulose membrane is sequentially provided with a T1 detection line coated with anti-calprotectin monoclonal antibody, a T2 detection line coated with anti-calprotectin monoclonal antibody, and a quality control line C coated with goat anti-chicken IgY antibody and / or goat anti-mouse IgG antibody.

[0065] The binding pad is immobilized with a colloidal gold-labeled calprotectin monoclonal antibody.

[0066] The sample diluent consists of the following components:

[0067]

[0068] The remainder is purified water, and the pH of the sample dilution is 7.0–7.6.

[0069] Furthermore, the detection threshold for the T1 test line is 50 ug / g. That is, when the fecal sample contains 50 ug or more of calprotectin, the T1 test line will appear, indicating that the patient may have a risk of active gastrointestinal inflammatory disease.

[0070] Furthermore, the detection threshold for the T2 test line is 200 ug / g. That is, when the amount of calprotectin in each gram of fecal sample is greater than or equal to 200 ug, the T2 test line will appear. The appearance of the T2 test line indicates that the patient may have active gastrointestinal inflammation, or a risk of relapse during clinical remission, or a graft rejection or transplant rejection reaction.

[0071] Furthermore, the interval between the T2 detection line and the T1 detection line is 4-6 mm, and the interval between the T2 detection line and the quality control line C is 4-6 mm.

[0072] Furthermore, the T1 detection line is coated with one or more of mouse anti-calprotectin monoclonal antibodies, goat anti-calprotectin monoclonal antibodies, and rabbit anti-calprotectin monoclonal antibodies. The coating concentration of the T1 detection line is 1.0–2.5 mg / ml, and the coating buffer is 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0073] Furthermore, the T2 detection line is coated with one or more of mouse anti-calprotectin monoclonal antibodies, goat anti-calprotectin monoclonal antibodies, and rabbit anti-calprotectin monoclonal antibodies. The coating concentration of the T2 detection line is 0.5–1.5 mg / ml, and the coating buffer is 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0074] Furthermore, the coating concentration of control line C was 1.0–2.0 mg / ml, and the coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0075] Furthermore, one end of the absorbent paper is pressed against one end of the nitrocellulose membrane at a distance of 1–2 mm, and / or

[0076] The absorbent paper is made of either glass fiber film or polyester film, preferably polyester film.

[0077] Furthermore, one end of the binding pad is pressed against the other end of the nitrocellulose membrane at a distance of 1–2 mm, and / or

[0078] The bonding pad is made of one of the following materials: glass fiber membrane, polyester membrane, or non-woven fabric, with glass fiber membrane being preferred.

[0079] Furthermore, the pad is immobilized with one or more of the following: mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody, and rabbit anti-calprotectin monoclonal antibody, and / or

[0080] The concentration of colloidal gold-labeled calprotectin monoclonal antibody is 10 μg / ml to 30 μg / ml, and / or

[0081] The particle size of colloidal gold is 20–50 nm.

[0082] Furthermore, the preparation process of the bonding pad is as follows:

[0083] 1) Monoclonal antibodies labeled with calprotectin:

[0084] Adjust the pH of the colloidal gold to 8.0–8.5 using 0.2M K₂CO₃ solution. Add calprotectin monoclonal antibody at a labeling concentration of 10–30 μg / ml. Stir and mix thoroughly for 30–60 min. Block with 20% BSA solution to a final blocking concentration of 0.5%–1%. Centrifuge and discard the supernatant. Reconstitute the precipitate with a reconstitution solution to half its original volume.

[0085] The reconstitution solution is a 0.02M Tris-HCl buffer solution containing 0.1-0.5% PEG20000, 0.5-1% casein, 1%-5% sucrose, and 0.05%-0.2% Tween 80, with a pH of 8.0-8.5.

[0086] 2) Using a gold sprayer, uniformly spray the gold-labeled calprotectin monoclonal antibody obtained in step 1) onto a glass fiber membrane, polyester membrane, or non-woven fabric. The gold spraying amount is 4ul / cm to 8ul / cm. Then, place it in a drying room at 37°C for 24 to 32 hours to dry.

[0087] The sample pad is placed at one end of the conjugate pad, and the other end of the sample pad is pressed against one end of the conjugate pad. The sample pad is pretreated with a sample pad treatment solution, which makes it better suited for fecal sample testing. The relevant components in the sample pad treatment solution help the sample pad to wet quickly, increase the hydrophilicity of the sample pad, effectively filter out and retain large molecular impurities and non-specific interfering substances in fecal samples, and effectively improve the detection accuracy of the test strip.

[0088] Furthermore, the sample pad is pretreated with a sample pad treatment solution, which consists of the following components:

[0089]

[0090]

[0091] The remainder is purified water, and the pH value of the sample pad treatment solution is 8.0 to 8.2.

[0092] In one embodiment: A test kit for detecting calprotectin in human feces comprises a sample diluent and a test strip. The test strip consists of a base plate 1, a nitrocellulose membrane 2, a conjugate pad 4, a sample pad 5, and absorbent paper 3. The absorbent paper 3, the nitrocellulose membrane 2, the conjugate pad 4, and the sample pad 5 are sequentially overlapped on the base plate 1. The test strip is further provided with an outer shell, which has an observation window and a sample application hole. The position of the observation window is adapted to the position of the nitrocellulose membrane 2, and the position of the sample application hole is adapted to the position of the sample pad 5.

[0093] The nitrocellulose membrane 2 is sequentially provided with a T1 detection line 21 coated with anti-calprotectin monoclonal antibody, a T2 detection line 22 coated with anti-calprotectin monoclonal antibody, and a quality control line C 23 coated with goat anti-chicken IgY antibody;

[0094] The binding pad 4 is immobilized with a colloidal gold-labeled calprotectin monoclonal antibody.

[0095] The sample diluent consists of the following components:

[0096]

[0097] The remainder is purified water, and the pH of the sample dilution is 7.0.

[0098] Furthermore, the detection threshold of the T1 detection line 21 is 50 ug / g.

[0099] Furthermore, the detection threshold of the T2 detection line 22 is 200 ug / g.

[0100] Furthermore, the interval between the T2 detection line 22 and the T1 detection line 21 is 4mm, and the interval between the T2 detection line 22 and the quality control line C 23 is 4mm.

[0101] Furthermore, the T1 detection line 21 was coated with mouse anti-calprotectin monoclonal antibody at a concentration of 1.0 mg / ml. The coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0102] Furthermore, the T2 detection line 22 was coated with mouse anti-calprotectin monoclonal antibody at a concentration of 0.5 mg / ml. The coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0103] Furthermore, the coating concentration of control line C 23 was 1.0 mg / ml, and the coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0104] Furthermore, one end of the absorbent paper 3 is pressed 1 mm away from one end of the nitrocellulose membrane 2, and / or

[0105] The absorbent paper 3 is made of glass fiber film.

[0106] Furthermore, one end of the bonding pad 4 is pressed 1 mm away from the other end of the nitrocellulose membrane 2, and / or

[0107] The material of bonding pad 4 is a glass fiber membrane.

[0108] Furthermore, the mat 4 was immobilized with a colloidal gold-labeled mouse anti-calprotectin monoclonal antibody, and / or

[0109] The concentration of colloidal gold-labeled calprotectin monoclonal antibody was 10 μg / ml, and / or

[0110] The particle size of colloidal gold is 20 nm.

[0111] Furthermore, the preparation process of pad 4 is as follows:

[0112] 1) Monoclonal antibodies labeled with calprotectin:

[0113] The pH of the colloidal gold was adjusted to 8.0 using 0.2M K₂CO₃ solution. Mouse calprotectin monoclonal antibody was added at a labeling concentration of 10 μg / ml. The mixture was stirred and reacted for 30 min. The mixture was then blocked with 20% BSA solution to a final blocking concentration of 0.5%. The supernatant was discarded by centrifugation, and the precipitate was reconstituted with a reconstitution solution to half its original volume.

[0114] The reconstitution solution is a 0.02M Tris-HCl buffer solution containing 0.1% PEG20000, 0.5% casein, 1% sucrose, and 0.05% Tween 80, with a pH of 8.0.

[0115] 2) Use a gold sprayer to uniformly spray the gold-labeled calprotectin monoclonal antibody obtained in step 1) onto the glass fiber membrane at a gold spraying amount of 4 μL / cm, and then place it in a drying room at 37°C for 24 hours.

[0116] Furthermore, the sample pad 5 is pretreated with a sample pad treatment solution, which consists of the following components:

[0117]

[0118] The remainder is purified water, and the pH value of the sample pad treatment solution is 8.0.

[0119] In another embodiment: A test kit for detecting calprotectin in human feces comprises a sample diluent and a test strip. The test strip consists of a base plate 1, a nitrocellulose membrane 2, a conjugate pad 4, a sample pad 5, and absorbent paper 3. The absorbent paper 3, the nitrocellulose membrane 2, the conjugate pad 4, and the sample pad 5 are sequentially overlapped on the base plate 1. The test strip is further provided with an outer shell, which has an observation window and a sample application hole. The position of the observation window is adapted to the position of the nitrocellulose membrane 2, and the position of the sample application hole is adapted to the position of the sample pad 5.

[0120] The nitrocellulose membrane 2 is sequentially provided with a T1 detection line 21 coated with anti-calprotectin monoclonal antibody, a T2 detection line 22 coated with anti-calprotectin monoclonal antibody, and a quality control line C 23 coated with goat anti-chicken IgY antibody;

[0121] The binding pad 4 is immobilized with a colloidal gold-labeled calprotectin monoclonal antibody.

[0122] The sample diluent consists of the following components:

[0123]

[0124]

[0125] The remainder is purified water, and the pH of the sample dilution is 7.6.

[0126] Furthermore, the detection threshold of the T1 detection line 21 is 50 ug / g.

[0127] Furthermore, the detection threshold of the T2 detection line 22 is 200 ug / g.

[0128] Furthermore, the interval between the T2 detection line 22 and the T1 detection line 21 is 6mm, and the interval between the T2 detection line 22 and the quality control line C 23 is 6mm.

[0129] Furthermore, the T1 detection line 21 was coated with goat anti-calprotectin monoclonal antibody at a concentration of 2.5 mg / ml. The coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0130] Furthermore, the T2 detection line 22 was coated with goat anti-calprotectin monoclonal antibody at a concentration of 1.5 mg / ml, and the coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0131] Furthermore, the coating concentration of control line C 23 was 2.0 mg / ml, and the coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0132] Furthermore, one end of the absorbent paper 3 is pressed against one end of the nitrocellulose membrane 2 at a distance of 2 mm, and / or

[0133] The absorbent paper 3 is made of polyester film.

[0134] Furthermore, one end of the bonding pad 4 is pressed 2 mm down the other end of the nitrocellulose membrane 2, and / or

[0135] The material of bonding pad 4 is polyester film.

[0136] Furthermore, the colloidal gold-labeled goat anti-calprotectin monoclonal antibody was immobilized on pad 4, and / or

[0137] The concentration of colloidal gold-labeled calprotectin monoclonal antibody was 30 μg / ml, and / or

[0138] The particle size of colloidal gold is 50 nm.

[0139] Furthermore, the preparation process of pad 4 is as follows:

[0140] 1) Monoclonal antibodies labeled with calprotectin:

[0141] The pH of the colloidal gold was adjusted to 8.5 using 0.2M K₂CO₃ solution. Goat calprotectin monoclonal antibody was added at a labeling concentration of 30 μg / ml. The mixture was stirred and reacted for 60 min. The mixture was then blocked with 20% BSA solution to a final blocking concentration of 1%. The supernatant was discarded by centrifugation, and the precipitate was reconstituted with a reconstitution solution to half its original volume.

[0142] The reconstitution solution is a 0.02 M Tris-HCl buffer solution containing 0.5% PEG20000, 1% casein, 5% sucrose, and 0.2% Tween 80, with a pH of 8.5.

[0143] 2) Use a gold sprayer to uniformly spray the gold-labeled calprotectin monoclonal antibody obtained in step 1) onto the polyester film at a gold spraying amount of 8 μL / cm, and then place it in a drying room at 37°C for 32 hours.

[0144] Furthermore, the sample pad 5 is pretreated with a sample pad treatment solution, which consists of the following components:

[0145]

[0146] The remainder is purified water, and the pH value of the sample pad treatment solution is 8.2.

[0147] In another embodiment: a test kit for detecting calprotectin in human feces comprises a sample diluent and a test strip. The test strip consists of a base plate 1, a nitrocellulose membrane 2, a conjugate pad 4, a sample pad 5, and absorbent paper 3. The absorbent paper 3, the nitrocellulose membrane 2, the conjugate pad 4, and the sample pad 5 are sequentially overlapped on the base plate 1. The test strip is further provided with an outer shell, which has an observation window and a sample application hole. The position of the observation window is adapted to the position of the nitrocellulose membrane 2, and the position of the sample application hole is adapted to the position of the sample pad 5, wherein:

[0148] The nitrocellulose membrane 2 is sequentially provided with a T1 detection line 21 coated with anti-calprotectin monoclonal antibody, a T2 detection line 22 coated with anti-calprotectin monoclonal antibody, and a quality control line C 23 coated with goat anti-mouse IgG antibody;

[0149] The binding pad 4 is immobilized with a colloidal gold-labeled calprotectin monoclonal antibody.

[0150] The sample diluent consists of the following components:

[0151] Sodium chloride 1.0%;

[0152] EDTA 0.06%;

[0153] Proclin-300 0.1%;

[0154] The remainder is purified water, and the pH of the sample dilution is 7.2.

[0155] Furthermore, the detection threshold of the T1 detection line 21 is 50 ug / g.

[0156] Furthermore, the detection threshold of the T2 detection line 22 is 200 ug / g.

[0157] Furthermore, the interval between the T2 detection line 22 and the T1 detection line 21 is 5mm, and the interval between the T2 detection line 22 and the quality control line C 23 is 5mm.

[0158] Furthermore, the T1 detection line 21 was coated with rabbit anti-calprotectin monoclonal antibody at a concentration of 2 mg / ml. The coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0159] In other embodiments, the T1 detection line 21 is coated with mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody and rabbit anti-calprotectin monoclonal antibody. The coating concentration of the T1 detection line 21 is 1.5 mg / ml, and the coating buffer is 0.01M PBS + 1% sucrose with a pH of 7.4.

[0160] Furthermore, the T2 detection line 22 was coated with rabbit anti-calprotectin monoclonal antibody at a concentration of 1 mg / ml. The coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0161] In other embodiments, the T2 detection line 22 is coated with mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody and rabbit anti-calprotectin monoclonal antibody. The coating concentration of the T2 detection line 22 is 1.2 mg / ml, and the coating buffer is 0.01M PBS + 1% sucrose with a pH of 7.4.

[0162] Furthermore, the coating concentration of control line C 23 was 1.5 mg / ml, and the coating buffer was 0.01 M PBS + 1% sucrose with a pH of 7.4.

[0163] Furthermore, one end of the absorbent paper 3 is pressed against one end of the nitrocellulose membrane 2 at a distance of 1.5 mm, and / or

[0164] The absorbent paper 3 is made of polyester film.

[0165] Furthermore, one end of the bonding pad 4 is pressed against the other end of the nitrocellulose membrane 2 at a distance of 1.5 mm, and / or

[0166] The material of the bonding pad 4 is non-woven fabric.

[0167] Furthermore, the rabbit anti-calprotectin monoclonal antibody immobilized with colloidal gold on pad 4, and / or

[0168] The concentration of colloidal gold-labeled calprotectin monoclonal antibody was 20 μg / ml, and / or

[0169] The particle size of colloidal gold is 30 nm.

[0170] In other embodiments, pad 4 is immobilized with colloidal gold-labeled mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody, rabbit anti-calprotectin monoclonal antibody, and / or

[0171] The concentration of colloidal gold-labeled calprotectin monoclonal antibody was 15 μg / ml, and / or

[0172] The particle size of colloidal gold is 40 nm.

[0173] Furthermore, the preparation process of pad 4 is as follows:

[0174] 1) Monoclonal antibodies labeled with calprotectin:

[0175] The pH of the colloidal gold was adjusted to 8.2 using 0.2M K₂CO₃ solution. Rabbit calprotectin monoclonal antibody was added at a labeling concentration of 20 μg / ml. The mixture was stirred and reacted for 45 min. The mixture was then blocked with 20% BSA solution to a final blocking concentration of 0.7%. The supernatant was discarded by centrifugation, and the precipitate was reconstituted with a reconstitution solution to half its original volume.

[0176] The reconstitution solution is a 0.02 M Tris-HCl buffer solution containing 0.3% PEG20000, 0.7% casein, 3% sucrose, and 0.1% Tween 80, with a pH of 8.3.

[0177] 2) Use a gold sprayer to uniformly spray the gold-labeled calprotectin monoclonal antibody obtained in step 1) onto the nonwoven fabric. The gold spraying amount is 6ul / cm. Then place it in a drying room at 37℃ for 28 hours to dry.

[0178] Furthermore, the sample pad 5 is pretreated with a sample pad treatment solution, which consists of the following components:

[0179]

[0180] The remainder is purified water, and the pH value of the sample pad treatment solution is 8.1.

[0181] The operational steps for detecting calprotectin in feces using the kit disclosed in this invention are as follows:

[0182] 1) Collect feces from multiple points using a feces collection stick (preferably covering the spiral groove at the far end of the collection device) (approximately 50 mg), then place it back into the feces collection container, tighten the cap, and mix thoroughly with the sample diluent.

[0183] 2) Tear open the aluminum foil bag, take out the reagent card and place it flat on the table;

[0184] 3) Unscrew the top cap of the sample collector and add 3 drops (approximately 90-100 μL) of the pretreated sample solution into the sample well of the test strip;

[0185] 4) Read the results through the observation window at 10-15 minutes. The results are invalid after 15 minutes.

[0186] Result interpretation:

[0187] a. Figure 2 This is an illustration of a negative test result, such as... Figure 2 As shown, a red band appears at control line C, and no bands are observed at test lines T1 and T2. The results indicate that the calprotectin content in the sample is not higher than 50 ug / g, there is no active gastrointestinal inflammation, no risk of recurrence (Crohn's disease or ulcerative colitis), and no transplant rejection reaction.

[0188] b. Figure 3 This is an illustration of a positive test result, such as... Figure 3 As shown, a red band appears at control line C, and only the T1 test line (pink or red) appears in the detection area. There is no band at the T2 test line. The results indicate that the content of calprotectin in the sample is greater than or equal to 50 ug / g and less than 200 ug / g, which may indicate the risk of active gastrointestinal inflammatory disease.

[0189] c. Figure 4 This is an illustration of a positive test result, such as... Figure 4 As shown, a red band appears at control line C, and the T1 and T2 test lines in the detection area are also visible. The results indicate that the content of calprotectin in the sample is greater than or equal to 200 ug / g, which suggests the possible presence of active gastrointestinal inflammation, risk of relapse during clinical remission, and graft rejection or transplant rejection.

[0190] d. All other cases are invalid results, such as Figure 5 As shown.

[0191] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A detection kit for calprotectin in human feces, characterized in that, The test strip consists of a sample diluent and a test paper. The test strip comprises a base plate, a nitrocellulose membrane, a conjugate pad, a sample pad, and absorbent paper. The absorbent paper, the nitrocellulose membrane, the conjugate pad, and the sample pad are sequentially overlapped on the base plate. The test strip also has an outer shell with an observation window and a sample application hole. The observation window is positioned to match the position of the nitrocellulose membrane, and the sample application hole is positioned to match the position of the sample pad. The nitrocellulose membrane is sequentially provided with a T1 detection line coated with anti-calprotectin monoclonal antibody, a T2 detection line coated with anti-calprotectin monoclonal antibody, and a quality control line C coated with goat anti-chicken IgY antibody and / or goat anti-mouse IgG antibody. The binding pad is immobilized with a colloidal gold-labeled calprotectin monoclonal antibody. The sample diluent consists of the following components: The remainder is purified water, and the pH of the sample dilution is 7.0–7.

6.

2. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, The detection threshold of the T1 detection line is 50 ug / g, and / or The detection threshold of the T2 detection line is 200 ug / g.

3. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, The interval between the T2 detection line and the T1 detection line is 4-6 mm, and the interval between the T2 detection line and the quality control line C is 4-6 mm.

4. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, The T1 test line is coated with one or more of the following: mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody, and rabbit anti-calprotectin monoclonal antibody. The coating concentration of the T1 test line is 1.0–2.5 mg / ml, and the coating buffer is 0.01M PBS + 1% sucrose, pH 7.4, and / or The T2 detection line is coated with one or more of the following: mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody, and rabbit anti-calprotectin monoclonal antibody. The coating concentration of the T2 detection line is 0.5–1.5 mg / ml, and the coating buffer is 0.01 M PBS + 1% sucrose, pH 7.4, and / or The coating concentration of quality control line C is 1.0–2.0 mg / ml, and the coating buffer is 0.01 M PBS + 1% sucrose with a pH of 7.

4.

5. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, One end of the absorbent paper is pressed against one end of the nitrocellulose membrane at a distance of 1–2 mm, and / or The absorbent paper is made of either glass fiber film or polyester film.

6. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, One end of the conjugate pad is pressed against the other end of the nitrocellulose membrane 1–2 mm away, and / or The bonding pad is made of one of the following materials: glass fiber membrane, polyester membrane, or non-woven fabric, with glass fiber membrane being preferred.

7. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, The binding pad is immobilized with one or more of the following: mouse anti-calprotectin monoclonal antibody, goat anti-calprotectin monoclonal antibody, and rabbit anti-calprotectin monoclonal antibody, and / or The concentration of colloidal gold-labeled calprotectin monoclonal antibody is 10 μg / ml to 30 μg / ml, and / or The particle size of colloidal gold is 20–50 nm.

8. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, The preparation process of the conjugate pad is as follows: 1) Monoclonal antibodies labeled with calprotectin: Adjust the pH of the colloidal gold to 8.0–8.5 using 0.2M K₂CO₃ solution. Add calprotectin monoclonal antibody at a labeling concentration of 10–30 μg / ml. Stir and mix thoroughly for 30–60 min. Block with 20% BSA solution to a final blocking concentration of 0.5%–1%. Centrifuge and discard the supernatant. Reconstitute the precipitate with a reconstitution solution to half its original volume. The reconstitution solution is a 0.02M Tris-HCl buffer solution containing 0.1-0.5% PEG20000, 0.5-1% casein, 1%-5% sucrose, and 0.05%-0.2% Tween 80, with a pH of 8.0-8.

5. 2) Using a gold sprayer, uniformly spray the gold-labeled calprotectin monoclonal antibody obtained in step 1) onto a glass fiber membrane, polyester membrane, or non-woven fabric. The gold spraying amount is 4ul / cm to 8ul / cm. Then, place it in a drying room at 37°C for 24 to 32 hours to dry.

9. The detection kit for detecting calprotectin in human feces as described in claim 1, characterized in that, The sample pad is pretreated with a sample pad treatment solution, which consists of the following components: Sodium chloride 0.05–0.1%; The remainder is purified water, and the pH value of the sample pad treatment solution is 8.0 to 8.2.

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