Colorectal cancer related serum metabolite joint detection kit and application thereof

Abstract

A colorectal cancer related serum metabolite joint detection kit comprises a rectangular flat box body and four pieces of chromatography test paper, the test paper is arranged at intervals in the left-right direction, and each piece of test paper is formed by sequentially connecting a sample pad, a combination pad, a nitrocellulose membrane detection pad and an absorption pad. A detection line and a quality control line are arranged on the detection pad, the detection line is respectively coated with fluorescein-coated antibodies of lactic acid, 5 (S)-hydroxyeicosatetraenoic acid, lysophosphatidylcholine and diacylglycerol, and the quality control line is coated with streptomycete antibiotic protein-HRP. The kit body is provided with a serum sample adding hole and an observation window, so that sample loading and result reading are facilitated. By optimizing the antibody coating amount (1.7-2.3 [mu] g / cm < 2 >), the detection sensitivity and specificity are ensured, the color is developed only when the concentration of the target metabolite exceeds a threshold value, and the method has the characteristics of high accuracy and rapid detection.

Description

Technical Field

[0001] The present invention relates to a combined detection kit for colorectal cancer-related serum metabolites and application thereof. Background Art

[0002] Colorectal cancer (CRC) is a malignant tumor that has become the third leading cause of cancer-related death worldwide due to multiple factors such as obesity, unhealthy diet, environmental pollution, alcoholism and lack of exercise. Studies have shown that the survival rate of CRC patients depends largely on early diagnosis; it is reported that the survival rate of TNM stage I CRC patients is as high as 92%, while the survival rate of stage IV CRC patients is only 10%. However, due to the lack of specific clinical manifestations, early CRC is usually difficult to detect.

[0003] For the detection method of this disease, see Chinese patent CN202211352611.3. Colonoscopy is the gold standard for diagnosing CRC, but due to its invasiveness, it is difficult to promote as a screening method. In contrast, non-invasive screening based on biomarkers has a high compliance rate, but the diagnostic effect of commonly used tumor biomarkers is not satisfactory. Therefore, it is of great significance to explore and develop new biomarkers to provide new non-invasive screening methods for CRC. Malignant tumors are accompanied by complex metabolic changes, which are even called metabolic diseases. Therefore, tumor metabolites are expected to become new markers for early diagnosis of tumors. CRC has been shown to have unique metabolic phenotypes and key metabolic pathways, such as anaerobic glycolysis and tricarboxylic acid cycle; these metabolic biomarkers are not only used for early cancer screening, but also provide new targets for treatment based on important metabolic pathways, but there is a lack of kits for the detection of serum metabolites related to colorectal cancer. Summary of the invention

[0004] The first technical problem to be solved by the present invention is to provide a combined detection kit for serum metabolites related to colorectal cancer. This kit can quickly detect relevant indicators at the same time by combining multiple specific antibodies related to colorectal cancer, thereby meeting the needs of qualitative and quantitative analysis.

[0005] The second technical problem to be solved by the present invention is to provide an application of the above-mentioned colorectal cancer-related serum metabolite combined detection kit in a product for detecting colon cancer-related serum metabolite indicators.

[0006] To solve the first technical problem mentioned above, the present invention provides a colorectal cancer-related serum metabolite combined detection kit, comprising a rectangular flat box body arranged horizontally and having a front-to-back length direction, wherein four chromatographic test strips are arranged in the box body, and the four chromatographic test strips are arranged at intervals in the left-right direction in the box body, and each chromatographic test strip comprises a horizontally arranged long strip backing plate with a front-to-back length direction, and a sample pad, a binding pad, a detection pad and an absorption pad are arranged on the upper side of the backing plate from front to back in sequence, the rear part of the sample pad is lifted up and overlapped with the front part of the binding pad, the rear part of the binding pad is lifted up and overlapped with the front part of the detection pad, the front part of the absorption pad is lifted up and overlapped with the rear part of the quality control line, and the detection pad is made of nitrocellulose membrane, and the four The detection pad of the chromatographic test paper is provided with two detection lines and quality control lines arranged at intervals in front and behind. The quality control lines of the four chromatographic test papers are coated with streptavidin-HRP. The detection lines of the four chromatographic test papers are respectively coated with four different preset serum metabolites related to colorectal cancer, namely lactic acid, 5(S)-hydroxyeicosatetraenoic acid, lysophosphatidylcholine and diacylglycerol. A serum loading hole is provided on the upper side of the box body corresponding to the sample pad of each chromatographic test paper, and the serum loading hole runs through the upper side of the box body in the vertical direction. An observation window is provided on the upper side of the box body corresponding to the detection pads of the four chromatographic test papers. The projection of the observation window in the vertical direction is sufficient to cover the detection lines and quality control lines of the four chromatographic test papers.

[0007] Preferably, the four preset antibodies are all fluorescein-coated antibodies.

[0008] Preferably, the coating amount of the preset antibodies on the four chromatographic test strips is 1.7-2.3 μg / cm 2 .

[0009] In order to solve the above-mentioned second technical problem, the present invention provides the use of a colorectal cancer-related serum metabolite combined detection kit in a product for detecting colon cancer-related serum metabolite indicators.

[0010] Advantages of the present invention: The detection kit provided by the present invention can be combined with lactic acid, 5(S)-hydroxyeicosatetraenoic acid, lysophosphatidylcholine and diacylglycerol antibodies to perform qualitative and quantitative analysis and detection of colorectal cancer-related indicators, and has the characteristics of high accuracy and rapid detection. In addition, it contains four highly correlated biomarker antibodies for colorectal cancer at one time, and can obtain the contents of four indicators at the same time in one test. The detection method is cheap and fast. The binding of antibodies to nitrocellulose membranes is better than that of polyclonal antibodies, so the selection of antibodies can improve their binding effect for accurate quantitative marking. The use of fluorescein encapsulation can allow the antibodies to have the effect of luminescence on a dark background under fluorescence microscopy, and can have better analytical effects for quantitative detection.

[0011] Working principle and detection limit control of preset antibodies:

[0012] 1. Working principle: Metabolites such as lactic acid act as antigens and combine with specific antibodies (such as lactic acid antibodies) on the test strip to form a complex and develop color.

[0013] Detection sensitivity: By adjusting the antibody coating amount (1.7~2.3μg / cm 2 ) controls the minimum detection limit to ensure that color is displayed only when the concentration of the target metabolite exceeds the threshold.

[0014] For example, a certain serum metabolite has a concentration range in normal human body, which is at a lower level, and its concentration is significantly increased in colorectal cancer patients. We set the antibody coating amount (i.e. the concentration of the antibody on the test strip), and the strip will only appear when the serum metabolite concentration exceeds the range and there is enough antigen to bind to it.

[0015] 2. Concentration determination:

[0016] Specific direction of change:

[0017] Lactic acid: The concentration in the serum of patients with colorectal cancer increases, and the serum of patients with colorectal cancer will show color.

[0018] 5(S)-Hydroxyeicosatetraenoic acid: Its concentration increases in the serum of patients with colorectal cancer, and the serum of patients with colorectal cancer will show color.

[0019] Lysophosphatidylcholine (lysoPC): Its concentration is elevated in the serum of patients with colorectal cancer, and the serum of patients with colorectal cancer will show color.

[0020] Diacylglycerol (DG): The concentration in the serum of patients with colorectal cancer is reduced, and the serum of patients with colorectal cancer does not show color. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the internal structure of the kit of the present invention (top view).

[0022] Figure 2 It is a top view of the kit of the present invention.

[0023] Figure 3 It is a schematic diagram of the structure of the chromatography test paper (side view).

[0024] Figure 4 and Figure 5 It is a schematic diagram of the screening and identification results of serum metabolites in the validation experiment.

[0025] Figure 6 This is a biomarker panel consisting of immune-related proteins validated by ELISA in an independent validation cohort in the validation experiment. DETAILED DESCRIPTION

[0026] See also Figure 1-Figure 3 A colorectal cancer-related serum metabolite combined detection kit comprises a rectangular flat box body 1 arranged horizontally and having a front-to-back length direction, four chromatographic test strips 2 are arranged in the box body 1, and the four chromatographic test strips 2 are arranged at intervals along the left and right directions in the box body 1, each chromatographic test strip 2 comprises a long strip backing plate 21 arranged horizontally and having a front-to-back length direction, and a sample pad 22, a binding pad 23, a detection pad 24 and an absorption pad 25 are arranged on the upper side of the backing plate 21 from front to back in sequence, the rear part of the sample pad 22 is lifted up and overlapped on the front part of the binding pad 23, the rear part of the binding pad 23 is lifted up and overlapped on the front part of the detection pad 24, the front part of the absorption pad 25 is lifted up and overlapped on the rear part of the quality control line 242, the detection pad 24 is made of nitrocellulose membrane, and the detection pads 24 of the four chromatographic test strips 2 are Each of the four chromatographic test strips 2 is provided with two detection lines 241 and a quality control line 242 arranged at intervals in front and behind. The quality control lines 242 of the four chromatographic test strips 2 are coated with streptavidin-HRP. The detection lines 241 of the four chromatographic test strips 2 are respectively coated with four different preset serum metabolites related to colorectal cancer, namely lactic acid, 5(S)-hydroxyeicosatetraenoic acid, hemolytic phosphatidylcholine and diacylglycerol. A serum sample injection hole 11 is provided at the upper side of the box body 1 corresponding to the sample pad 22 of each chromatographic test strip 2, and the serum sample injection hole 11 passes through the upper side of the box body 1 in the vertical direction. An observation window 12 is provided at the upper side of the box body 1 corresponding to the detection pads 24 of the four chromatographic test strips 2. The projection of the observation window 12 in the vertical direction is sufficient to cover the detection lines 241 and the quality control lines 242 of the four chromatographic test strips 2.

[0027] The four preset antibodies are all fluorescein-coated antibodies.

[0028] The coating amounts of the preset antibodies on the detection lines 241 of the four chromatography test strips 2 are detailed in Table 1.

[0029] Table 1

[0030]

[0031]

[0032] Verification experiment:

[0033] In this experiment, serum metabolites related to colorectal cancer were screened, and the abbreviations in the figure represent their meanings: Con (healthy control) and CRC (colorectal cancer).

[0034] 1. Non-targeted serum metabolomics analysis, the results of which are as follows Figure 4 and Figure 5 shown.

[0035] (1) 100 μL of serum sample was added to a 1.5 mL centrifuge tube, and 400 μL of a solution containing 0.02 mg / mL internal standard (L-2-chlorophenylalanine) (acetonitrile: methanol = 1:1 (v:v)) was added to extract metabolites. The tube was vortexed for 30 s to mix, sonicated at low temperature for 30 min (5°C, 40 kHz), and placed at -20°C for 30 min to precipitate proteins. The tube was then centrifuged for 15 min (4°C, 13,000 g), the supernatant was discarded, and the tube was dried with nitrogen.

[0036] (2) The sample was re-dissolved with 100 μL of a solution (acetonitrile: water = 1:1) and extracted by low-temperature ultrasound for 5 minutes (5°C, 40 kHz), followed by centrifugation at 13000 g and 4°C for 10 minutes. The supernatant was transferred to a sample vial for LC-MS / MS analysis.

[0037] (3) LC-MS / MS analysis of samples was performed using a Thermo UHPLC-Q Exactive HF-X system equipped with an ACQUITY HSS T3 column (100 mm × 2.1 mm id, 1.8 μm; Waters, USA).

[0038] (4) The mobile phase was 0.1% formic acid aqueous solution: acetonitrile (95:5, v / v) (solvent A) and 0.1% formic acid aqueous solution: acetonitrile: isopropanol: water (47.5:47.5, v / v) (solvent B). The flow rate was 0.40 mL / min, and the column temperature was 40°C. The injection volume was 3 μL.

[0039] (5) Progenesis QI software was used to process the raw data of the mass spectrometer and convert it into a common format. Metabolites were identified by comparing it with the metabolite database (HMDB, Metlin, Majorbio Database). Then, principal component analysis (PCA) and orthogonal least partial squares discriminant analysis (OPLS-DA) were performed using R language. Based on the variable importance in projection (VIP) obtained by the OPLS-DA model and the p value obtained by the T test, metabolites with VIP>1 and p<0.05 were identified as significantly differential metabolites.

[0040] Figure 4 The heat map shows the differential serum metabolites between CRC and control groups. Figure 5 The following are the four differentially abundant metabolites between the CRC group and the control group (Con) and their diagnostic values ​​for CRC. The substances with higher diagnostic values ​​include the following serum metabolites:

[0041] 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), lactic acid and lysophosphatidylcholine (lysoPC) (0:0 / 16:0), diacylglycerol (DG) (8:0 / 10:0 / 0:0).

[0042] 2. Screen the above serum metabolite biomarkers and verify them using ELISA. The results are as follows: Figure 6 shown.

[0043] (1) Add control serum and colorectal cancer serum to a 96-well plate pre-coated with capture antibody.

[0044] (2) Wash five times with detergent.

[0045] (3) Add human detection antibodies (lactic acid, 5(S)-hydroxyeicosatetraenoic acid (5(S)-HETE), lysophosphatidylcholine (lysoPC) and diacylglycerol (DG)) and incubate at 37°C for 1 hour.

[0046] (4) Wash five times with detergent.

[0047] (5) Add streptavidin-HRP and incubate at 37°C for 30 minutes.

[0048] (6) Add substrate and incubate at room temperature for 15 minutes.

[0049] (7) Add stop solution to terminate the reaction.

[0050] (8) Place the 96-well plate on a microplate reader, read the optical density at 450 nm, and calculate the level of serum metabolites in each test sample based on the standard curve.

[0051] (9) After normalization by serum volume, all serum metabolite concentrations were converted to μg / mL.

[0052] After the above experiments, it was found that four biomarkers (lactic acid, 5(S)-hydroxyeicosatetraenoic acid, lysophosphatidylcholine and diacylglycerol) were highly correlated with colorectal cancer and could be used as screening indicators for joint screening of colorectal cancer.

[0053] Because the four biomarkers (lactic acid, 5(S)-hydroxyeicosatetraenoic acid, lysophosphatidylcholine and diacylglycerol) are highly correlated with colorectal cancer, making them into a combined detection immunoassay kit can help to quickly and accurately detect the levels of colorectal cancer-related serum metabolite indicators.

[0054] In the above embodiments, the antibodies are derived from:

[0055] Lactic acid: anti-lactic acid-BSA conjugate antibody, manufacturer: BioVision, catalog number: K628-100;

[0056] 5(S)-HETE: anti-5(S)-HETE antibody, manufacturer: Cayman Chemical, catalog number: 10010585;

[0057] Lysophosphatidylcholine: Anti-LysoPC polyclonal antibody, manufacturer: Aviva Systems Biology, catalog number: OAAB10201;

[0058] Diacylglycerol: Anti-DAG kinase antibody, Manufacturer: Cayman Chemical, Cat. No.: 10009933.

Claims

1. A combined detection kit for serum metabolites related to colorectal cancer, characterized in that: The invention comprises a rectangular flat box body which is horizontally arranged and has a front-to-back length direction, wherein four chromatographic test strips are arranged in the box body, and the four chromatographic test strips are arranged in a left-right spaced relationship in the box body, and each chromatographic test strip comprises a long strip backing plate which is horizontally arranged and has a front-to-back length direction, and a sample pad, a binding pad, a detection pad and an absorption pad are arranged on the upper side of the backing plate from front to back in sequence, the rear part of the sample pad is lifted upward and overlapped on the front part of the binding pad, the rear part of the binding pad is lifted upward and overlapped on the front part of the detection pad, the front part of the absorption pad is lifted upward and overlapped on the rear part of the quality control line, the detection pad is made of nitrocellulose membrane, and the detection pads of the four chromatographic test strips are provided with two front-to-back spacing cloths. The detection lines and quality control lines of the four chromatographic test strips are coated with streptavidin-HRP, and the detection lines of the four chromatographic test strips are respectively coated with four different preset antibodies for serum metabolites related to colorectal cancer, namely lactic acid, 5S-hydroxyeicosatetraenoic acid, lysophosphatidylcholine and diacylglycerol. A serum loading hole is provided at the upper side of the box body corresponding to the sample pad of each chromatographic test strip, and the observation window is provided at the upper side of the box body corresponding to the detection pads of the four chromatographic test strips. The projection of the observation window along the vertical direction is sufficient to cover the detection lines and quality control lines of the four chromatographic test strips.

2. A colorectal cancer-related serum metabolite combined detection kit according to claim 1, characterized in that: The four preset antibodies are all fluorescein-coated antibodies.

3. A colorectal cancer-related serum metabolite combined detection kit according to claim 1, characterized in that: The coating amount of the preset antibodies on the four chromatographic test strips is 1.7-2.3 μg / cm 2 .

4. Use of the colorectal cancer-related serum metabolite combined detection kit according to any one of claims 1 to 3 in a product for detecting colon cancer-related serum metabolite indicators.

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