阅读说明：本技术 一种快速检测肝病的多联检试剂盒及制备方法 (Multi-joint detection kit for rapidly detecting liver diseases and preparation method thereof ) 是由 刘万里 吴棉涛 汪艳涛 潘秀华 于 2019-04-28 设计创作，主要内容包括：本发明公开了一种快速检测肝病的多联检试剂盒及制备方法,采用荧光免疫层析法,将检测肝病的四个标志物：分别为血管生成素样蛋白2(angptl2)、甲胎蛋白(AFP)、血清骨桥蛋白(OPN)及人类软骨糖蛋白(YKL-40)制备成四种荧光检测卡,四种检测卡平行排列连接在一起,组成试剂盒本体,其中每相邻的两个检测卡有一个加样孔,且每个检测卡上均设置有读卡窗。本发明可同时检测肝病的4种不同标志物,可以对肝硬化、肝癌及肝癌预后全面监测,使肝病的早期检测更加快速准确；操作简单、检测时间短,只需要加样2次,即可得到4种检测结果,加样完成后,10min即可出结果,更适合基层应用。(The invention discloses a multi-joint detection kit for rapidly detecting liver diseases and a preparation method thereof, wherein a fluorescence immunochromatography method is adopted to detect four markers of liver diseases: four fluorescence detection cards are respectively prepared from angiopoietin-like protein 2(angptl2), alpha-fetoprotein (AFP), serum Osteopontin (OPN) and human cartilage glycoprotein (YKL-40), the four detection cards are arranged in parallel and connected together to form a kit body, each two adjacent detection cards are provided with a sample adding hole, and each detection card is provided with a card reading window. The invention can simultaneously detect 4 different markers of liver diseases, can comprehensively monitor liver cirrhosis, liver cancer and liver cancer prognosis, and enables the early detection of the liver diseases to be faster and more accurate; the method is simple to operate and short in detection time, 4 detection results can be obtained only by adding samples for 2 times, and after the samples are added, the results can be obtained in 10min, so that the method is more suitable for basic application.)

1. A multiple examination kit for rapidly detecting liver diseases is characterized in that a fluorescence immunochromatography method is adopted, and four markers for detecting liver diseases are as follows: four fluorescence detection cards are respectively prepared from angiopoietin-like protein 2(angptl2), alpha-fetoprotein (AFP), serum Osteopontin (OPN) and human cartilage glycoprotein (YKL-40), the four detection cards are arranged in parallel and connected together to form a kit body, each two adjacent detection cards are provided with a sample adding hole, and each detection card is provided with a card reading window.

2. The multiple diagnostic kit for rapid detection of liver disease as claimed in claim 1, wherein the fluorescent marker used in the fluoroimmunoassay is one of lanthanide chelates of europium, samarium and dysprosium.

3. The multiple test kit for rapid detection of liver diseases according to claim 1, wherein the angiopoietin-like protein 2(angptl2) test card comprises a quality control line C and a detection line T.

4. The multiple diagnostic kit for rapid detection of liver disease as claimed in claim 1, wherein the alpha-fetoprotein (AFP) detection card comprises a quality control line C and a detection line T.

5. A multiple test kit for rapid detection of liver diseases according to claim 1, wherein the serum Osteopontin (OPN) test card comprises a quality control line C, a test line T1 and a test line T2, wherein the line T1 represents the b-subtype of serum Osteopontin (OPN) and the line T2 represents the C-subtype of serum Osteopontin (OPN).

6. The multiple diagnostic kit for rapid detection of liver disease as claimed in claim 1, wherein the human cartilage glycoprotein (YKL-40) detection card comprises a quality control line C and a detection line T.

7. A multi-unit test kit for rapid detection of liver diseases as claimed in claim 1, wherein each test card has a test paper therein, the test paper comprises a bottom plate, and a sample pad, a fluorescent microsphere labeling pad, a coating film and a water absorption pad are sequentially connected to the bottom plate.

8. A method for preparing a multiple test kit for rapid detection of liver diseases according to any one of claims 1 to 7, comprising the steps of:

s1: preparation of sample pad

Cutting the sample pad into size of 20mm × 300mm, soaking in sample pad buffer solution, taking out after 1 hr, and drying at room temperature for 16-18 hr;

s2: preparation of fluorescent microsphere labeling pad

1) Preparation of fluorescent microsphere label pad of angiopoietin-like protein 2(angptl2)

① pretreatment of microsphere, adding 10ul of europium-containing fluorescent microsphere with particle size of 300nm into 0.1M MES buffer solution (pH 5.5) 1ml, and mixing;

② activating microsphere by adding 20mg/ml EDC50ul into the above solution, mixing, and reacting in a rotary incubator for 20 min;

③ quenching the microspheres, namely adding 2-mercaptoethanol with the final concentration of 0.02M to quench the redundant EDC;

④ coupling antibody, adding angiopoietin-like protein 2(angptl2) monoclonal antibody 35ug into the solution, mixing uniformly on a rotary incubator, and reacting for 1 h;

⑤ sealing microsphere, adding 3% liquid gelatin 200ul, sealing, and mixing on rotary culture device for 30 min;

⑥ purifying the microspheres by separating the protein microsphere complex from the solution with desalting column;

⑦ storing, adding 300ul of storage solution into desalting column, transferring to EP tube, and ultrasonic scattering for 2-4 times;

⑧ spraying the obtained solution onto glass cellulose membrane with gold spraying and membrane scratching instrument at a spraying amount of 1-5 μ L/cm, placing into oven at 37 deg.C, and drying for 6-8 hr to obtain fluorescent microsphere labeling pad of angiopoietin-like protein 2(angptl 2);

2) replacing the angiopoietin-like protein 2(angptl2) antibody with an alpha-fetoprotein (AFP) antibody and a human cartilage glycoprotein (YKL-40) antibody according to the method to prepare an alpha-fetoprotein (AFP) fluorescent microsphere label pad and a human cartilage glycoprotein (YKL-40) fluorescent microsphere label pad respectively;

3) preparation of serum Osteopontin (OPN) fluorescent microsphere label pad

① pretreatment of microsphere, adding 10ul of europium-containing fluorescent microsphere with particle size of 300nm into 0.1M MES buffer solution (pH 5.5) 1ml, and mixing;

② activating microsphere by adding 20mg/ml EDC50ul into the above solution, mixing, and reacting in rotary incubator for 20 min;

③ quenching the microspheres, namely adding 2-mercaptoethanol with the final concentration of 0.02M to quench the redundant EDC;

④ coupling antibody, adding serum Osteopontin (OPN) b subtype antibody and serum Osteopontin (OPN) c subtype antibody into the above solution 30ug respectively, mixing uniformly on rotary incubator, and reacting for 1 h;

⑤ sealing microsphere, adding 3% liquid gelatin 200ul, sealing, and mixing on rotary culture device for 30 min;

⑥ purifying the microspheres by separating the protein microsphere complex from the solution with desalting column;

⑦ storing, adding 300ul of storage solution into desalting column, transferring to EP tube, and ultrasonic scattering for 2-4 times;

⑧ spraying the obtained solution onto glass cellulose membrane with gold spraying and membrane scratching instrument at a spraying amount of 1-5 μ L/cm, placing into oven at 37 deg.C, and drying for 6-8 hr to obtain serum Osteopontin (OPN) fluorescent microsphere labeled pad;

s3: preparation of coating film

1) Preparation of angiopoietin-like protein 2(angptl2) coating film

Detection line (T line): marking a line C and a line T on the nitrocellulose membrane, marking the line C and the line T by using a marker pen, wherein the distance between the line C and the line T is 0.5cm, the distance between the line T and the lower edge of the nitrocellulose membrane is 1cm, and coating the angiopoietin-like protein 2(angptl2) monoclonal antibody by diluting the monoclonal antibody to 1.0mg/ml by using 2% methanol, 1% sucrose and 0.01M PBS (pH7.4); the scribing concentration is 1.5 mu L/cm, and the speed is 100 mm/s;

quality control line (line C): coating goat anti-mouse IgG polyclonal antibody diluted to 0.8mg/ml with 3% methanol, 1% sucrose, 0.01M PBS (pH7.4) at a streaking concentration of 1.5. mu.L/cm and a speed of 100 mm/s;

putting the nitrocellulose membrane coated with the C line and the T line into a 37 ℃ oven, and drying for 6-8 hours to obtain an angiopoietin-like protein 2(angptl2) coated membrane;

2) the preparation method of the alpha-fetoprotein (AFP) and human cartilage glycoprotein (YKL-40) coating is the same as that of the angiopoietin-like protein 2(angptl 2);

3) preparation of serum Osteopontin (OPN) coating film

Detection line (T1 and T2 lines): marking a line on the nitrocellulose membrane, marking a line C, a line T1 and a line T2 at one end of the membrane by using a marker pen, wherein the distance between the line C and the line T1 is 0.4cm, the distance between the line T1 and the line T2 is 0.4cm, the distance between the line T2 and the lower edge of the nitrocellulose membrane is 0.9cm, and coating the antibody of the b subtype serum Osteopontin (OPN) to 1.0mg/ml by using 2% methanol, 1% sucrose and 0.01M PBS (pH7.4) to obtain a line T1; coating serum Osteopontin (OPN) subtype c antibody diluted to 1.0mg/ml with 2% methanol, 1% sucrose, 0.01M PBS, pH7.4 as line T2; the scribing concentration is 1.5 mu L/cm, and the speed is 100 mm/s;

quality control line (line C): coating goat anti-mouse IgG polyclonal antibody diluted to 0.8mg/ml with 3% methanol, 1% sucrose, 0.01M PBS (pH7.4) at a streaking concentration of 1.5. mu.L/cm and a speed of 100 mm/s;

putting the nitrocellulose membrane coated with the C line, the T1 line and the T2 line into a 37 ℃ oven, and drying for 6-8 hours to obtain a serum Osteopontin (OPN) coating membrane;

s4: preparation of test paper

Sequentially overlapping and assembling the sample pad, the fluorescent microsphere marking pad, the coating film and the water absorption pad, wherein the water absorption pad and the fluorescent microsphere marking pad are respectively overlapped at two ends of the coating film, and a detection area is formed on the surface of the coating film; overlapping and pressing the sample pad on the fluorescent microsphere marking pad, cutting the sample pad into 4mm wide after assembly to obtain test paper, and preparing four test paper strips according to different fluorescent microsphere marking pads and coating films;

s5: preparation detection box

And (4) sequentially mounting the four test strips obtained in the step (S4) on a bottom cover of the detection card, covering a box cover, wherein the position of a card reading window in the box cover corresponds to the detection area of the envelope film, the positions of the sample adding holes are positioned in the middle of the sample pads, and two adjacent sample pads share one sample adding hole, so that the multi-joint detection box is manufactured.

9. The method for preparing a multiple diagnostic kit for rapid detection of liver diseases according to claim 8, wherein the formulation of the buffer solution in the sample pad is as follows: 2% BSA, 1% PEG4000, 0.5% Tween were dissolved in 0.01M PBS (pH7.4) buffer.

10. The method for preparing a multiplex assay kit for the rapid detection of liver diseases as claimed in claim 8, wherein the preservation solution is 0.05M glycine-NaOH buffer solution with pH8.5 containing 1% casein sodium, 5% trehalose, 1% PVP, 0.5% EDTA.

Technical Field

The invention relates to the technical field of disease detection, in particular to a multi-test kit for rapidly detecting liver diseases and a preparation method thereof.

Background

At present, about 20 hundred million liver disease virus infectors and 3.5 million liver disease virus carriers are existed in the world, wherein 1.3 million liver disease virus carriers exist in China, 3000 million chronic hepatitis B patients exist, and thus, 1 liver disease virus carrier exists in almost every 10 Chinese people. 50 million new cases of liver diseases occur in China every year, about 28 million patients die of liver disease related diseases every year, and about 35 million patients die of liver cirrhosis and liver cancer caused by various liver diseases. Thus, liver disease has become one of the serious health hazards. The later stage of liver disease is easy to develop into liver cancer, the most effective method for treating liver cancer at present is surgical resection and liver transplantation, but most patients have a middle and late stage of treatment, and the surgical resection rate is only 10% -30%, so that the early diagnosis of liver cancer is very important and is the key of clinical diagnosis and prognosis.

Currently, the diagnosis of liver cancer mainly depends on serological examination and imaging diagnosis. The serological detection mainly detects tumor markers in serum, the currently mainly detected marker is Alpha Fetoprotein (AFP), but the sensitivity of the Alpha Fetoprotein (AFP) in small liver cancer is only about 40 percent, namely, the occurrence and the development of tumors cannot be accurately diagnosed by singly using one tumor marker, the detection accuracy can be improved by jointly detecting a plurality of markers, and missed diagnosis and false positive are avoided. The marker combination with more diagnostic value is selected, the sensitivity and specificity of the detection of the existing marker are improved, and the method is very important for early diagnosis of liver cancer.

Disclosure of Invention

The invention aims to provide a multi-joint detection kit for rapidly detecting liver diseases and a preparation method thereof, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme:

a multiple examination kit for rapidly detecting liver diseases adopts a fluorescence immunochromatography method to detect four markers of liver diseases: four fluorescence detection cards are respectively prepared from angiopoietin-like protein 2(angptl2), alpha-fetoprotein (AFP), serum Osteopontin (OPN) and human cartilage glycoprotein (YKL-40), the four detection cards are arranged in parallel and connected together to form a kit body, each two adjacent detection cards are provided with a sample adding hole, and each detection card is provided with a card reading window.

As a further scheme of the invention: the fluorescent marker adopted in the fluorescence immunochromatography is one of lanthanide chelates of europium, samarium and dysprosium.

As a still further scheme of the invention: the angiopoietin-like protein 2(angptl2) detection card comprises a quality control line C and a detection line T.

As a still further scheme of the invention: the Alpha Fetoprotein (AFP) detection card comprises a quality control line C and a detection line T.

As a still further scheme of the invention: the serum Osteopontin (OPN) detection card comprises a quality control line C, a detection line T1 and a detection line T2, wherein the T1 line represents a serum Osteopontin (OPN) b subtype, and the T2 line represents a serum Osteopontin (OPN) C subtype.

As a still further scheme of the invention: the human cartilage glycoprotein (YKL-40) detection card comprises a quality control line C line and a detection line T line.

As a still further scheme of the invention: each detection card is internally provided with detection test paper, the test paper comprises a bottom plate, and a sample pad, a fluorescent microsphere marking pad, a coating film and a water absorption pad are sequentially connected on the bottom plate.

The invention also provides a preparation method of the multiple examination reagent box for liver diseases, which sequentially comprises the following steps:

s1: preparation of sample pad

Cutting the sample pad into size of 20mm × 300mm, soaking in sample pad buffer solution, taking out after 1 hr, and drying at room temperature for 16-18 hr;

s2: preparation of fluorescent microsphere labeling pad

1) Preparation of fluorescent microsphere label pad of angiopoietin-like protein 2(angptl2)

① pretreatment of microsphere, adding 10ul of europium-containing fluorescent microsphere with particle size of 300nm into 0.1M MES buffer solution (pH 5.5) 1ml, and mixing;

② activating microsphere by adding 20mg/ml EDC50ul into the above solution, mixing, and reacting in a rotary incubator for 20 min;

③ quenching the microspheres, namely adding 2-mercaptoethanol with the final concentration of 0.02M to quench the redundant EDC;

④ coupling antibody, adding angiopoietin-like protein 2(angptl2) monoclonal antibody 35ug into the solution, mixing uniformly on a rotary incubator, and reacting for 1 h;

⑤ sealing microsphere, adding 3% liquid gelatin 200ul, sealing, and mixing on rotary culture device for 30 min;

⑥ purifying the microspheres by separating the protein microsphere complex from the solution with desalting column;

⑦ storing, adding 300ul of storage solution into desalting column, transferring to EP tube, and ultrasonic scattering for 2-4 times;

⑧ spraying the obtained solution onto glass cellulose membrane with gold spraying and membrane scratching instrument at a spraying amount of 1-5 μ L/cm, placing into oven at 37 deg.C, and drying for 6-8 hr to obtain fluorescent microsphere labeling pad of angiopoietin-like protein 2(angptl 2);

2) replacing the angiopoietin-like protein 2(angptl2) antibody with an alpha-fetoprotein (AFP) antibody and a human cartilage glycoprotein (YKL-40) antibody according to the method to prepare an alpha-fetoprotein (AFP) fluorescent microsphere label pad and a human cartilage glycoprotein (YKL-40) fluorescent microsphere label pad respectively;

3) preparation of serum Osteopontin (OPN) fluorescent microsphere label pad

① pretreatment of microsphere, adding 10ul of europium-containing fluorescent microsphere with particle size of 300nm into 0.1M MES buffer solution (pH 5.5) 1ml, and mixing;

② activating microsphere by adding 20mg/ml EDC50ul into the above solution, mixing, and reacting in rotary incubator for 20 min;

③ quenching the microspheres, namely adding 2-mercaptoethanol with the final concentration of 0.02M to quench the redundant EDC;

④ coupling antibody, adding serum Osteopontin (OPN) b subtype antibody and serum Osteopontin (OPN) c subtype antibody into the above solution 30ug respectively, mixing uniformly on rotary incubator, and reacting for 1 h;

⑤ sealing microsphere, adding 3% liquid gelatin 200ul, sealing, and mixing on rotary culture device for 30 min;

⑥ purifying the microspheres by separating the protein microsphere complex from the solution with desalting column;

⑦ storing, adding 300ul of storage solution into desalting column, transferring to EP tube, and ultrasonic scattering for 2-4 times;

⑧ spraying the obtained solution onto glass cellulose membrane with gold spraying and membrane scratching instrument at a spraying amount of 1-5 μ L/cm, placing into oven at 37 deg.C, and drying for 6-8 hr to obtain serum Osteopontin (OPN) fluorescent microsphere labeled pad;

s3: preparation of coating film

1) Preparation of angiopoietin-like protein 2(angptl2) coating film

Detection line (T line): marking a line C and a line T on the nitrocellulose membrane, marking the line C and the line T by using a marker pen, wherein the distance between the line C and the line T is 0.5cm, the distance between the line T and the lower edge of the nitrocellulose membrane is 1cm, and coating the angiopoietin-like protein 2(angptl2) monoclonal antibody by diluting the monoclonal antibody to 1.0mg/ml by using 2% methanol, 1% sucrose and 0.01M PBS (pH7.4); the scribing concentration is 1.5 mu L/cm, and the speed is 100 mm/s;

quality control line (line C): coating goat anti-mouse IgG polyclonal antibody diluted to 0.8mg/ml with 3% methanol, 1% sucrose, 0.01M PBS (pH7.4) at a streaking concentration of 1.5. mu.L/cm and a speed of 100 mm/s;

putting the nitrocellulose membrane coated with the C line and the T line into a 37 ℃ oven, and drying for 6-8 hours to obtain an angiopoietin-like protein 2(angptl2) coated membrane;

2) the preparation method of the alpha-fetoprotein (AFP) and human cartilage glycoprotein (YKL-40) coating is the same as that of the angiopoietin-like protein 2(angptl 2);

3) preparation of serum Osteopontin (OPN) coating film

Detection line (T1 and T2 lines): marking a line on the nitrocellulose membrane, marking a line C, a line T1 and a line T2 at one end of the membrane by using a marker pen, wherein the distance between the line C and the line T1 is 0.4cm, the distance between the line T1 and the line T2 is 0.4cm, the distance between the line T2 and the lower edge of the nitrocellulose membrane is 0.9cm, and coating the antibody of the b subtype serum Osteopontin (OPN) to 1.0mg/ml by using 2% methanol, 1% sucrose and 0.01M PBS (pH7.4) to obtain a line T1; coating serum Osteopontin (OPN) subtype c antibody diluted to 1.0mg/ml with 2% methanol, 1% sucrose, 0.01M PBS, pH7.4 as line T2; the scribing concentration is 1.5 mu L/cm, and the speed is 100 mm/s;

quality control line (line C): coating goat anti-mouse IgG polyclonal antibody diluted to 0.8mg/ml with 3% methanol, 1% sucrose, 0.01M PBS (pH7.4) at a streaking concentration of 1.5. mu.L/cm and a speed of 100 mm/s;

putting the nitrocellulose membrane coated with the C line, the T1 line and the T2 line into a 37 ℃ oven, and drying for 6-8 hours to obtain a serum Osteopontin (OPN) coating membrane;

s4: preparation of test paper

Sequentially overlapping and assembling the sample pad, the fluorescent microsphere marking pad, the coating film and the water absorption pad, wherein the water absorption pad and the fluorescent microsphere marking pad are respectively overlapped at two ends of the coating film, and a detection area is formed on the surface of the coating film; overlapping and pressing the sample pad on the fluorescent microsphere marking pad, cutting the sample pad into 4mm wide after assembly to obtain test paper, and preparing four test paper strips according to different fluorescent microsphere marking pads and coating films;

s5: preparation detection box

And (4) sequentially mounting the four test strips obtained in the step (S4) on a bottom cover of the detection card, covering a box cover, wherein the position of a card reading window in the box cover corresponds to the detection area of the envelope film, the positions of the sample adding holes are positioned in the middle of the sample pads, and two adjacent sample pads share one sample adding hole, so that the multi-joint detection box is manufactured.

As a further scheme of the invention: the buffer formulation in the sample pad is as follows: 2% BSA, 1% PEG4000, 0.5% Tween were dissolved in 0.01M PBS (pH7.4) buffer.

As a still further scheme of the invention: the preservation solution is 0.05M glycine-NaOH buffer solution with the pH value of 8.5, and contains 1% sodium caseinate, 5% trehalose, 1% PVP and 0.5% EDTA.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention can simultaneously detect 4 different markers of liver diseases, can comprehensively monitor liver cirrhosis, liver cancer and liver cancer prognosis, enables the early detection of the liver diseases to be faster and more accurate, and does not have a multi-detection reagent for detecting the liver diseases in the market at present.

2. The method is simple to operate and short in detection time, 4 detection results can be obtained only by adding samples for 2 times, and the results can be obtained in 10min after the sample addition is finished, so that the method is more suitable for basic application.

3. The invention adopts a time-resolved fluorescent microsphere labeling technology, the detection card has high sensitivity, wide linear range and accurate result, better meets the clinical application, and is provided with the OPN detection test strip capable of simultaneously detecting the subtype b and the subtype c.

Drawings

Fig. 1 is a schematic structural view of a multiple examination kit for rapidly detecting liver diseases.

In the figure: 1-kit body, 2-detection card, 3-card reading window and 4-sample adding hole.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The terms referred to in the present invention are specifically as follows:

angiopoietin-like protein 2(angptl 2): angiopoietin-like protein 2(angptl2) is one member of the angiopoietin-like white family, and regulates the deposition and degradation of extracellular matrix by increasing the expression and activity of metalloproteinases. By detecting the content of angiopoietin-like protein 2(angptl2), the fibrosis degree of the liver disease population can be predicted.

Alpha-fetoprotein (AFP): alpha-fetoprotein (AFP) is a specific clinical index for diagnosing primary liver cancer. The content of AFP in the serum of normal human is not less than 20 mug/L, AFP is increased in about 70% of primary liver cancer patients, and is increased 6-12 months before symptoms appear, but about 30% of liver cancer patients are normal Alpha Fetoprotein (AFP).

Serum Osteopontin (OPN): serum Osteopontin (OPN) is widely distributed in various tissues and cells, the expression of the normal human serum Osteopontin (OPN) is very slight, but the expression of the OPN is high in highly invasive tumors, and the OPN is used as a liver cancer tumor marker, plays an important role in the development and diagnosis of liver cancer, and can make up the defects of AFP in the diagnosis of the liver cancer.

Human cartilage glycoprotein (YKL-40): is one member of the mammalian chitinase family, and researches show that YKL-40 has certain correlation with the prognosis of patients with liver cancer, and the survival rate of patients with positive YKL-40 expression after operation is obviously reduced compared with that of negative patients.

Referring to fig. 1, in the embodiment of the present invention, a multiple-test kit for rapidly detecting liver diseases employs a fluorescence immunochromatography method to detect four markers of liver diseases: four fluorescence detection cards 2 are respectively prepared from angiopoietin-like protein 2(angptl2), alpha-fetoprotein (AFP), serum Osteopontin (OPN) and human cartilage glycoprotein (YKL-40), the four detection cards 2 are connected in parallel to form a kit body 1, each two adjacent detection cards 2 are provided with a sample adding hole 4, each detection card 2 is provided with a card reading window 3, and during operation, only two samples need to be added, and four detection results can be obtained within 10 min.

The fluorescent marker adopted in the fluorescence immunochromatography is one of lanthanide chelates of europium, samarium and dysprosium.

The angiopoietin-like protein 2(angptl2) detection card comprises a quality control line C and a detection line T.

The Alpha Fetoprotein (AFP) detection card comprises a quality control line C and a detection line T.

The serum Osteopontin (OPN) detection card comprises a quality control line C, a detection line T1 and a detection line T2, wherein the T1 line represents a serum Osteopontin (OPN) b subtype, and the T2 line represents a serum Osteopontin (OPN) C subtype.

The human cartilage glycoprotein (YKL-40) detection card comprises a quality control line C line and a detection line T line.

Each detection card 2 is internally provided with detection test paper which comprises a bottom plate, and a sample pad, a fluorescent microsphere marking pad, a coating film and a water absorption pad are sequentially connected on the bottom plate.

The latter technical scheme of the invention is to provide a preparation method of a multiple examination reagent box for liver diseases, which sequentially comprises the following steps:

s1: preparation of sample pad

1) Cutting the sample pad into size of 20mm × 300mm, soaking in sample pad buffer solution, taking out after 1 hr, and drying at room temperature for 16-18 hr;

2) the buffer formulation in the sample pad was as follows: 2% BSA, 1% PEG4000, 0.5% Tween in 0.01M PBS (pH7.4) buffer;

s2: preparation of fluorescent microsphere labeling pad

1) Preparation of fluorescent microsphere label pad of angiopoietin-like protein 2(angptl2)

① pretreatment of microsphere, adding 10ul of europium-containing fluorescent microsphere with particle size of 300nm into 0.1M MES buffer solution (pH 5.5) 1ml, and mixing;

② activating microsphere by adding 20mg/ml EDC50ul into the above solution, mixing, and reacting in a rotary incubator for 20 min;

③ quenching the microspheres, namely adding 2-mercaptoethanol with the final concentration of 0.02M to quench the redundant EDC;

④ coupling antibody, adding angiopoietin-like protein 2(angptl2) monoclonal antibody 35ug into the solution, mixing uniformly on a rotary incubator, and reacting for 1 h;

⑤ sealing microsphere, adding 3% liquid gelatin 200ul, sealing, and mixing on rotary culture device for 30 min;

⑥ purifying the microspheres by separating the protein microsphere complex from the solution with desalting column;

⑦ storing, adding 300ul of a storage solution into desalting column, transferring to EP tube, and ultrasonically scattering for 2-4 times, wherein the storage solution is 0.05M glycine-NaOH buffer solution with pH of 8.5 and containing 1% casein sodium, 5% trehalose, 1% PVP and 0.5% EDTA;

⑧ spraying the obtained solution onto glass cellulose membrane with gold spraying and membrane scratching instrument at a spraying amount of 1-5 μ L/cm, placing into oven at 37 deg.C, and drying for 6-8 hr to obtain fluorescent microsphere labeling pad of angiopoietin-like protein 2(angptl 2);

2) replacing the angiopoietin-like protein 2(angptl2) antibody with an alpha-fetoprotein (AFP) antibody and a human cartilage glycoprotein (YKL-40) antibody according to the method to prepare an alpha-fetoprotein (AFP) fluorescent microsphere label pad and a human cartilage glycoprotein (YKL-40) fluorescent microsphere label pad respectively;

3) preparation of serum Osteopontin (OPN) fluorescent microsphere label pad

① pretreatment of microsphere, adding 10ul of europium-containing fluorescent microsphere with particle size of 300nm into 0.1M MES buffer solution (pH 5.5) 1ml, and mixing;

② activating microsphere by adding 20mg/ml EDC50ul into the above solution, mixing, and reacting in rotary incubator for 20 min;

③ quenching the microspheres, namely adding 2-mercaptoethanol with the final concentration of 0.02M to quench the redundant EDC;

④ coupling antibody, adding serum Osteopontin (OPN) b subtype antibody and serum Osteopontin (OPN) c subtype antibody into the above solution 30ug respectively, mixing uniformly on rotary incubator, and reacting for 1 h;

⑤ sealing microsphere, adding 3% liquid gelatin 200ul, sealing, and mixing on rotary culture device for 30 min;

⑥ purifying the microspheres by separating the protein microsphere complex from the solution with desalting column;

⑦ storing, adding 300ul of a storage solution into desalting column, transferring to EP tube, and ultrasonically scattering for 2-4 times, wherein the storage solution is 0.05M glycine-NaOH buffer solution with pH of 8.5 and containing 1% casein sodium, 5% trehalose, 1% PVP and 0.5% EDTA;

⑧ spraying the obtained solution onto glass cellulose membrane with gold spraying and membrane scratching instrument at a spraying amount of 1-5 μ L/cm, placing into oven at 37 deg.C, and drying for 6-8 hr to obtain serum Osteopontin (OPN) fluorescent microsphere labeled pad;

s3: preparation of coating film

1) Preparation of angiopoietin-like protein 2(angptl2) coating film

Detection line (T line): marking a line C and a line T on the nitrocellulose membrane, marking the line C and the line T by using a marker pen, wherein the distance between the line C and the line T is 0.5cm, the distance between the line T and the lower edge of the nitrocellulose membrane is 1cm, and coating the angiopoietin-like protein 2(angptl2) monoclonal antibody by diluting the monoclonal antibody to 1.0mg/ml by using 2% methanol, 1% sucrose and 0.01M PBS (pH7.4); the scribing concentration is 1.5 mu L/cm, and the speed is 100 mm/s;

quality control line (line C): coating goat anti-mouse IgG polyclonal antibody diluted to 0.8mg/ml with 3% methanol, 1% sucrose, 0.01M PBS (pH7.4) at a streaking concentration of 1.5. mu.L/cm and a speed of 100 mm/s;

putting the nitrocellulose membrane coated with the C line and the T line into a 37 ℃ oven, and drying for 6-8 hours to obtain an angiopoietin-like protein 2(angptl2) coated membrane;

2) the preparation method of the alpha-fetoprotein (AFP) and human cartilage glycoprotein (YKL-40) coating is the same as that of the angiopoietin-like protein 2(angptl 2);

3) preparation of serum Osteopontin (OPN) coating film

Detection line (T1 and T2 lines): marking a line on the nitrocellulose membrane, marking a line C, a line T1 and a line T2 at one end of the membrane by using a marker pen, wherein the distance between the line C and the line T1 is 0.4cm, the distance between the line T1 and the line T2 is 0.4cm, the distance between the line T2 and the lower edge of the nitrocellulose membrane is 0.9cm, and coating the antibody of the b subtype serum Osteopontin (OPN) to 1.0mg/ml by using 2% methanol, 1% sucrose and 0.01M PBS (pH7.4) to obtain a line T1; coating serum Osteopontin (OPN) subtype c antibody diluted to 1.0mg/ml with 2% methanol, 1% sucrose, 0.01M PBS, pH7.4 as line T2; the scribing concentration is 1.5 mu L/cm, and the speed is 100 mm/s;

quality control line (line C): coating goat anti-mouse IgG polyclonal antibody diluted to 0.8mg/ml with 3% methanol, 1% sucrose, 0.01M PBS (pH7.4) at a streaking concentration of 1.5. mu.L/cm and a speed of 100 mm/s;

putting the nitrocellulose membrane coated with the C line, the T1 line and the T2 line into a 37 ℃ oven, and drying for 6-8 hours to obtain a serum Osteopontin (OPN) coating membrane;

s4: preparation of test paper

Sequentially overlapping and assembling the sample pad, the fluorescent microsphere marking pad, the coating film and the water absorption pad, wherein the water absorption pad and the fluorescent microsphere marking pad are respectively overlapped at two ends of the coating film, and a detection area is formed on the surface of the coating film; overlapping and pressing the sample pad on the fluorescent microsphere marking pad, cutting the sample pad into 4mm wide after assembly to obtain test paper, and preparing four test paper strips according to different fluorescent microsphere marking pads and coating films;

s5: preparation detection box

And (4) sequentially mounting the four test strips obtained in the step (S4) on a bottom cover of the detection card, and then covering a box cover, wherein the position of a card reading window 3 in the box cover corresponds to the detection area of the envelope film, the position of a sample adding hole 4 is positioned in the middle of the sample pad, and two adjacent sample pads share one sample adding hole 4, so that the multi-joint detection box is manufactured.

The specific application method of the invention is as follows:

1) when the invention is used, firstly, the instrument is opened, a chip with the same batch number as the reagent is inserted, the ID card is read, then 10 mul of sample is accurately absorbed and added into the diluent to be uniformly mixed for later use;

2) opening the package in the detection box, taking out the detection box, and horizontally placing the detection box;

3) sucking 75 mul of diluted sample, adding the sample into a sample adding hole of the detection box, and then starting timing;

4) selecting the sample type "plasma/serum" or "whole blood" by the instrument;

5) after the reaction is carried out for 10min at room temperature, clicking a 'test' button on the instrument, starting the detection of the instrument and displaying the result;

6) clicking 'printing' can print a detection result report.

The multi-examination kit for rapidly detecting liver diseases provided by the invention has the following advantages:

the invention can simultaneously detect 4 different markers of liver diseases, can comprehensively monitor liver cirrhosis, liver cancer and liver cancer prognosis, enables the early detection of the liver diseases to be faster and more accurate, and does not have a multi-detection reagent for detecting the liver diseases in the market at present.

The method is simple to operate and short in detection time, 4 detection results can be obtained only by adding samples for 2 times, and the results can be obtained in 10min after the sample addition is finished, so that the method is more suitable for basic application.

The invention adopts a time-resolved fluorescent microsphere labeling technology, the detection card has high sensitivity, wide linear range and accurate result, better meets the clinical application, and is provided with the OPN detection test strip capable of simultaneously detecting the subtype b and the subtype c.

The invention detects 1000 patients with liver diseases, and the result shows that: the sensitivity of single indicator detection of angptl2 is 80.37%, the specificity is 82.66%, the sensitivity of single indicator detection of AFP is 80.53%, the specificity is 81.29%, the sensitivity of single indicator detection of OPN is 81.36%, the specificity is 83.64%, the sensitivity of single indicator detection of YKL-4 is 82.72%, and the specificity is 83.51%;

the sensitivity of the dual-index detection of angptl2 and AFP is 84.45%, the specificity is 84.87%, the sensitivity of the dual-index detection of angptl2 and OPN is 84.01%, the specificity is 85.72%, the sensitivity of the dual-index detection of angptl2 and YKL-40 is 85.23%, the specificity is 84.33%, the sensitivity of the dual-index detection of AFP and OPN is 85.76%, the specificity is 86.12%, the sensitivity of the dual-index detection of AFP and YKL-40 is 84.97%, the specificity is 86.23%, the sensitivity of the dual-index detection of OPN and YKL-40 is 85.65%, and the specificity is 86.83%;

the detection sensitivity of three indexes of angptl2, AFP and OPN is 90.16%, the detection specificity is 91.48%, the detection sensitivity of three indexes of angptl2, AFP and YKL-40 is 89.67%, the detection specificity is 90.10%, the detection sensitivity of three indexes of angptl2, OPN and YKL-40 is 91.23%, the detection specificity is 92.88%, and the detection sensitivity of three indexes of AFP, OPN and YKL-40 is 89.55% and the detection specificity is 90.36%;

the sensitivity of detection of the four indicators of angptl2, AFP, OPN and YKL-40 is 95.15% and the specificity is 96.37%.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.