CN107688093B - Lung cancer detection test strip, kit and use method thereof - Google Patents

Abstract

The invention relates to the field of biological medical treatment, in particular to a lung cancer detection test strip, a lung cancer detection kit and a use method of the lung cancer detection test strip. The test strip comprises a sample absorption pad, a reaction membrane, a water absorption pad and a bottom plate; the sample absorbing pad is coated with a GM1 capture antibody, the GM1 capture antibody is labeled with an indicator for showing signal intensity; a detection area and a quality inspection area are arranged on the reaction membrane; the detection area is fixedly coated with a GM1 detection agent, and the GM1 detection agent is a GM1 detection antibody or a GM-TAG protein with an amino acid sequence shown as SEQ ID NO. 1; and a secondary antibody corresponding to the GM1 capture antibody is fixed on the quality detection area. The detection tool provided by the invention is suitable for large-scale manufacturing, is convenient to use and is suitable for wide popularization; has the capability of early finding lung cancer and strong diagnosis reliability.

Description

Lung cancer detection test strip, kit and use method thereof

Technical Field

The invention relates to the field of biological medical treatment, in particular to a lung cancer detection test strip, a lung cancer detection kit and a use method of the lung cancer detection test strip.

Background

Lung Cancer (LC) is the most prominent cancer in the world, with approximately 130 million deaths each year. The national tumor registration center chenomenqing, equal to 2016, 1, 25 published 2015 chinese Cancer statistics in the CA Cancer J Clin journal. There were 429.2 new cases of cancer and 281.4 deaths reported in 2015 in china. Lung cancer is the most common cancer and is also the leading cause of cancer death.

The health risks and deaths associated with lung cancer may also increase due to a variety of environmental changes, including aging. Lung cancer can be divided into a number of sub-categories, depending on cytological and cellular origin. The major types include non-small cell carcinoma (NSCLC) and small cell carcinoma (SCLC).

Despite advances in radiology, surgery, and chemotherapy, the 5-year survival rate of lung cancer has remained essentially unchanged for the past 30 years, with only 15% of patients surviving 5 years or more after initial diagnosis. These low survival rates are mainly due to the fact that some concurrent disorders prevent early diagnosis, limiting the efficacy of therapeutic interventions.

Current low-throughput diagnostic methods include sputum smears, chest X-rays, and CT scans. These methods are not suitable for high throughput screening of a large population only when the patient has specific symptoms, such as long-term cough, expectoration, hemoptysis, discomfort, pain or weight loss, and visits the hospital to a doctor. In addition, these methods have a high false positive and must be matched with subsequent invasive means, such as bronchoscopy or tissue biopsy, for definitive diagnosis. Cytological examinations are based on cell morphology, structure and anatomy, and can be processed using hematoxylin and eosin staining. This approach is widely used for medical diagnosis and is the gold standard for cancer screening biopsy. Hematoxylin stains the nucleus of the cell, which is then counterstained with a solution of eosin Y, which stains cytoplasmic components, typically including intracellular and extracellular proteins. The staining method can be used for in situ determination of key oncogenic proteins; such as Epidermal Growth Factor Receptor (EGFR), cyclin D or K-Kas. Ligases associated with these antibodies to anti-cancer proteins, such as horseradish peroxidase (HRP) or Alkaline Phosphatase (AP) or fluorescent dyes such as fluorescein, can be used to find the amount or cellular location of any oncogene in the biopsy. Similarly, the use of tumor suppressor antibodies, such as p53 or retinoblastoma protein (pRB), can be used to find signs of weakness or altered cellular location; such methods may all suggest an increased likelihood of cancer. Although these methods are mature, they are slow and cannot be used for large-scale high-throughput screening.

Many methods are expected to more effectively diagnose lung cancer. For example, lung cancer is diagnosed using specific primers or probes for nucleic acids that can encode proteins, values of which are believed to be indicative of the status of the lung cancer (i.e., phosphatidylinositol-glycan-specific phospholipase D; GPLD 1). However, such a method using the protein as a biomarker for lung cancer diagnosis has not been commercialized yet. Since early lung cancer is scattered in different locations of the lung, it is difficult to detect early lung cancer by any method of detection using blood, which generally reflects the condition of the entire circulatory system. Therefore, there is a need to develop a low cost and efficient method for rapid diagnosis, and which is easy to use from time to time.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a lung cancer detection test strip, a kit containing the test strip and a using method of the test strip.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention relates to a lung cancer detection test strip, which comprises a sample absorption pad, a reaction membrane, a water absorption pad and a bottom plate;

the sample absorbing pad is coated with a GM1 capture antibody, the GM1 capture antibody is labeled with an indicator for showing signal intensity;

a detection area and a quality inspection area are arranged on the reaction membrane;

the detection area is fixedly coated with a GM1 detection agent, and the GM1 detection agent is a GM1 detection antibody or a GM-TAG protein with an amino acid sequence shown as SEQ ID NO. 1;

and a secondary antibody corresponding to the GM1 capture antibody is fixed on the quality detection area.

The test strip provided by the invention is used for quickly and conveniently diagnosing lung cancer. Based on the applicant's previous scientific studies, the applicant used sputum (mucus expectorated from the lower respiratory tract, especially the trachea and bronchi) for diagnosis. This makes the detection simple and easy. Previous chinese-english studies by the applicant have identified several chemical biomarkers that allow the method of the present invention to diagnose lung cancer more quickly and economically than other methods, without patient discomfort. Based on these biomarkers, applicants developed tools for diagnosis.

In our previous studies, the mass spectrometer platform could easily build a database from sputum samples and analysis of these data could discover clinically relevant data. Using principal component analysis and high content analysis methods, healthy control groups can be distinguished from those patients who require further lung cancer examinations. Although not all of these patients are subsequently identified as lung cancer patients, those suspected lung cancer patients have symptoms that are considered by physicians to be associated with cancer and should be classified as "unhealthy". To further identify biomarkers that distinguish between cancer and non-cancer, more sophisticated data mining tools are required. The ROC curve is a widely used tool for determining the effect of biomarkers. The ROC curve shows the relationship between true positive and false positive, which can determine the effect of using a metabolite. The area under the ROC curve can represent the classification performance of one parameter: lung and non-lung cancer. By using ROCCET analysis, applicants determined a series of LTQ-MS-metabolites whose AUC values were greater than 0.8, a threshold for identifying the utility of the substance. These metabolites are present in relatively high concentrations in cancer sputum samples.

GM1(monosialoganglioside) is a ganglioside containing one sialic acid, also known as monosialotetrahexosyl ganglioside, also known as sialylglycosphingolipid, with a molecular weight of 1574. GM1 is primarily used for neural tissue studies, but it can be found in most cell types that they are involved in cell-cell recognition, cell-matrix attachment, cell growth and cell differentiation. Applicants have found that increased concentrations of glycosphingolipids in sputum are a key biomarker for early detection of lung cancer with accuracy rates above 80%. Other similar applications exist, such as the detection of small cell carcinomas in tissue biopsies of patients fused to hemocyanin.

An important feature of glycosphingolipids is that it binds to motifs within certain toxins produced by certain bacterial pathogens, such as the B subunit of cholera toxin. This correlates with the pathogenicity of cholera, but the GM1 binding site can dissociate from the rest of the B subunit protein and lose its pathogenic effect. Similarly, enterotoxigenic escherichia coli (ETEC), an important factor in morbidity and mortality worldwide, remains. The major virulence factor released by ETEC is heat labile enterotoxin LT, which is similar in structure and function to cholera toxin. LTB binds to glycosphingolipids, the host receptor for toxins, but interactions with type a blood glucose and e.

Applicants have discovered through studies that the protein GM-TAG binds GM1 with high affinity, and it should be noted that this protein has a histidine TAG at the C-terminus (shown in SEQ ID NO: 1), which means that its isolation is economical.

The poly-histidine-tagged recombinant protein can be conveniently expressed in E.coli. Bacteria can be harvested by centrifugation and the resulting cell pellet lysed to release cellular proteins. After incubation in an affinity resin containing cobalt ions, the labeled proteins are separated and the polyhistidine tag binds with high affinity. The resin is then washed to remove non-interacting proteins. The GM-TAG may then be further applied as described below.

Preferably, in the lung cancer test strip as described above, the indicator for indicating signal intensity includes any one of a fluorescent substance, a quantum dot, a digoxin-labeled probe, biotin, a radioisotope, an electron-dense substance, colloidal gold, or an enzyme.

Preferably, the test strip for detecting lung cancer as described above, wherein the fluorescent substance comprises Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein, 5-carboxy-2 ', 4', 5 ', 7' -tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7, 6-FAM, dansyl chloride, fluorescein, HEX, 6-JOE, NBD (7-nitrobenzo-2-oxa-1, 3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresyl fast violet, cresyl Blue violet, brilliant cresol Blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinyl fluorescein, rare earth metal cryptate, tripyridyldiamine europium, europium cryptate, diamine, bispyanin, La Jolla Blue dye, allophycocyanin, allocyanin B, phycocyanin C, phycocyanin R, thiamine, phycoerythrin R, REG, rhodamine Green, rhodamine isothiocyanate, rhodamine red, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and texas red.

Preferably, the test strip for detecting lung cancer as described above, wherein the radioisotope includes¹¹⁰In、¹¹¹In、¹⁷⁷Lu、¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁹⁰Y、⁸⁹Zr、⁹⁴mTc、⁹⁴Tc、⁹⁹mTc、¹²⁰I、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、^154-158Gd、³²P、¹¹C、¹³N、¹⁵O、¹⁸⁶Re、¹⁸⁸Re、⁵¹Mn、⁵²mMn、⁵⁵Co、⁷²As、⁷⁵Br、⁷⁶Br、⁸²mRb and⁸³sr.

Preferably, the lung cancer test strip as described above, the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase and glucose oxidase.

Preferably, the lung cancer detection test strip is provided with a test paper holder outside.

More preferably, the test paper holds in the palm and includes casing and lower casing, go up the casing with the casing is airtight formation inner space in four sides with the mode of gomphosis down be equipped with the locating piece on the casing, the locating piece is arranged in proper order the middle part of casing down, the quantity of locating piece does test paper strip quantity adds 1, adjacent two distance between the locating piece with the width of test paper strip is the same, all sets up one in the space between two adjacent locating pieces of each test paper strip go up and be equipped with application of sample hole and observation window on the casing, the application of sample hole corresponds all the sample pad of test paper strip, the observation window corresponds all the detection line and the quality control line of test paper strip.

According to one aspect of the invention, the invention also relates to a kit containing the test strip.

Preferably, the kit as described above, further comprising a risk indicator strip;

the risk indication strip is recorded with the relationship between the different signal intensities displayed by the detection area and the risk of lung cancer.

The risk indicator strip has the same signal type as the detection area, for example, when the GM1 capture antibody is labeled with horseradish peroxidase, the reaction membranes of the detection area and the quality detection area can be simultaneously coated with a substrate TMB of the horseradish peroxidase, and because the TMB is a chromogen substrate of the horseradish peroxidase, when the reaction is positive, a chromogenic reaction occurs in the detection area and the quality detection area, and then the concentration of GM1 in a sample to be detected is different, and the color concentration is different, and at the moment, the risk indicator strip can adopt a colorimetric card form to prompt the content of GM1 in the sample to be detected.

According to one aspect of the invention, the invention also relates to a method for detecting GM1 protein using a test strip as described above or a kit as described above, comprising:

and dropwise adding a sample to be detected on the sample absorption pad, detecting the signal intensity of the detection area on the premise that the quality detection area has a reaction signal after the sample absorption pad, the reaction membrane and the sample to be detected completely react, and quantifying according to the signal relation between the concentration of the GM1 protein and the signal intensity.

Preferably, the sample to be tested is sputum or saliva, as described above.

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

1) the detection tool provided by the invention is suitable for large-scale manufacturing;

2) the detection tool provided by the invention is easy to use, can be used as a daily screening means, and can be popularized and used in a large scale;

3) the detection tool provided by the invention has the capacity of early detecting lung cancer;

4) the detection tool provided by the invention has higher diagnosis reliability of more than 80%.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of an embodiment of example 1; GM1 is abbreviated herein as GM;

FIG. 2 is a schematic diagram of an embodiment of example 2; GM1 is abbreviated herein as GM.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

The tool detects saliva or sputum. It uses an adsorption strip to which antibody ("free" GM1 capture antibody, first dark color block from left in fig. 1) is attached. The antibody can capture GM1 and be labeled with a related enzyme for detection, typically horseradish peroxidase or alkaline phosphatase, which causes a color change. The left end of the strip is attached with dry free GM1 capture antibody, and the antibody can be diffused on the strip by capillary action by adding the solution of the test substance.

In a defined area, these antibodies were contacted with a detection zone (second dark color block from left in FIG. 1) and these GM1 capture antibodies were immobilized on the strip. The antibody in the detection zone has no associated enzyme and it recognizes a different epitope than the GM1 capture antibody, so it will bind to GM1, forming a sandwich region containing the immobilized GM1 detection antibody-GM 1- "free" GM1 capture antibody, and a detectable enzyme. This effectively concentrates the "free" GM1 capture abzyme complex to this portion of the adsorption strip.

The enzyme activity concentration can be represented by means of a color display by applying the assay substrate externally. Alternatively, the assay substrates may be attached to an absorbent strip, which may be dissolved and released to effect upon contact with the sample solution.

A positive control is important in the test, and is included in the test. An additional antibody, the secondary antibody to the GM1 capture antibody, was attached to a separate area of the strip, the quality control area (third dark area from left in fig. 1). This antibody bound to GM1 capture antibody, regardless of whether this GM1 capture antibody had bound to GM 1. Thus, if the GM1 capture antibody is cultured in rabbits, any rabbit antibody binding antibody cultured in mice, goats, or pigs may be used. Thus, free GM1 capture antibody will be concentrated to yet another region of the absorbent strip. Importantly, enzyme activity was shown in this green region regardless of the presence of GM1, and thus could be detected as described previously.

Thus, according to FIG. 1, a positive result is obtained if the detection zone and the quality control zone show color, and a negative result is obtained if only the quality control zone shows color. The depth of staining in the test area correlates with the number of GM1 and thus the risk of lung cancer. The correlation can be obtained by comparing the correlation with a prepared risk indication strip, and the risk indication strip is recorded with the relation between different color depths displayed by a detection area and the lung cancer risk.

Example 2

In this example, a novel test strip improved over example 1 is provided. The attached GM1 detection antibody was replaced by the attached GM-TAG protein. The binding sites of GM-TAG to the GM1 molecule are different from those of the "free" GM1 capture antibody. Therefore, there is no competition for the capture location of GM 1. This will enhance the sensitivity of the GM-TAG, GM1-GM1 capture antibody sandwich region. Otherwise, the two tools were identical, including positive controls and correlation of color intensity with GM1 content and lung cancer risk.

Examples of the experiments

This technique has been tested currently using samples from the uk and china respectively. The British sample consisted of 67 individuals, and 33 healthy control samples were included. The remaining 34 included lung and non-lung cancer patients. By using the method, the diagnosis accuracy of the lung cancer/non-lung cancer can reach 80 percent on the whole. The Chinese samples were 50 persons in total, including 15 healthy control samples. Using our method, lung/non-lung cancer diagnosis accuracy was also achieved at 80% level by comparison with confirmed markers. Notably, the diagnosis accuracy for small cell carcinoma reached 100% in these samples.

Case 1: a 45 year old male tells the physician that he is suffering from severe cough, shortness of breath, and fever. The physician finds that he smokes 20 cigarettes on average a day and asks him to provide a sputum sample, which is tested using the method of the invention. The doctor can make a definite diagnosis of the patient. Using this technique, the overall diagnostic period is less than 24 hours.

Case 2: a 55 year old patient was hospitalized for chest pain, cough, expectoration and a history of smoking. The physician asks her to provide a sputum sample and uses our method for detection. This method compares the compounds in the patient's sputum sample with markers we have found. Based on the comparison, the test identifies the patient as having stage 2 lung cancer. The physician then confirms the diagnosis and provides the corresponding treatment by other methods, including chest fluoroscopy, etc.

Case 3: a72 year old male, with a history of smoking, lost weight due to unknown causes, persistent coughing, and difficulty walking up stairs, went to a community hospital for examination. The physician asks him to provide a sputum sample. The lung cancer of the patient is detected by using the method, and is found to be positive by comparing with the marker. According to the detection result, the community doctor sends the patient to a special hospital for further examination, and confirms the examination result.

Case 4: a 79 year old female with a history of smoking was consulted with a physician for persistent coughing, spitting, and wheezing. The community doctor asks her to provide a sputum sample. The samples were compared to the markers using our method. The results showed lung cancer negative. To ensure that misdiagnoses are not made, community doctors send the patient to a special hospital department of respiration, which proves to be a problem with other respiratory systems, and perform corresponding treatments.

Case 5: an 84 year old patient was diagnosed with lung cancer. Due to recurrent symptoms, diagnosis is required. However, the potential harm and resulting morbidity and mortality associated with patients with these severe symptoms from traditional diagnostic means has not been able to make these diagnoses. To monitor the progress of the condition, the patient is asked to provide a sputum sample. By using the method, accurate detection results can be rapidly provided for doctors to refer to, and corresponding treatment methods can be formulated.

Case 6: a 77 year old female patient had a history of smoking, weight loss of unknown cause, asthma, and was unable to walk long distances to the community hospital for diagnosis. The community doctors collected the sputum sample and detected by using our method, and found that the patient is positive for lung cancer, so that the patient is submitted to a special hospital for targeted treatment.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Lihongjun, Hanjiwan, Luyi Shi, Aerjiade, mu (Mur, Luis Alejandro Jose)

<120> lung cancer detection test strip, kit and use method thereof

<160>1

<170>PatentIn version 3.3

<210>1

<211>449

<212>PRT

<213> Artificial sequence

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Met Arg Gly Ser Met Ala Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu

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Gln Asn Arg Lys Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu

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Val Ser Glu Glu Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp

35 40 45

Phe Lys Leu Gly Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr

50 55 60

Gln Asn Glu Asn Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile

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Ser Phe Trp Ile Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr

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Thr Ile Ile Asp Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile

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Ile Ser Asn Phe Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu

115 120125

Gln Ser Ile Asn Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr

130 135 140

Asn Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met

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Lys Ile Tyr Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu

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Leu Thr Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys

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Ile Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met

195 200 205

Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys Asp

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Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp

225 230 235 240

Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn

245 250 255

Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser Arg Gln Ile Val

260 265 270

Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu Gly Tyr Lys Ile

275 280285

Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn Asp Thr Arg Val Arg Gly

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Gly Asp Ile Leu Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn

305 310 315 320

Leu Phe Met Lys Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu

325 330 335

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His Asn Tyr Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu

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His

Claims (8)

1. The application of the test strip in the preparation of the lung cancer detection kit is characterized in that the test strip comprises a sample absorption pad, a reaction membrane, a water absorption pad and a bottom plate;

the sample absorbing pad is coated with a GM1 capture antibody, the GM1 capture antibody is labeled with an indicator for showing signal intensity;

a detection area and a quality inspection area are arranged on the reaction membrane;

the detection area is fixedly coated with a GM1 detection agent, and the GM1 detection agent is a GM-TAG protein with an amino acid sequence shown as SEQ ID NO. 1;

and a secondary antibody corresponding to the GM1 capture antibody is fixed on the quality detection area.

2. The use according to claim 1, wherein the indicator showing signal intensity comprises any one of a fluorescent substance, a digoxin-labeled probe, biotin, an electron-dense substance, colloidal gold, or an enzyme.

3. The use of claim 2, wherein said fluorescent substance comprises Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminoacridine, BODIPY 630/650, BODIPY650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5-carboxyrhodamine, 6-carboxytetramethylrhodamine, Cascade Blue, 6-FAM, dansyl chloride, HEX, 6-JOE, NBD (7-nitrobenz-2-oxa-1, 3-diazole), Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid, isophthalic acid, cresolfast violet, cresyl violet, brilliant cresol Blue, para-toluic acid, erythrosine, phthalocyanine, azomethine, cyanine, and/or mixtures thereof, Any one of xanthine, succinyl fluorescein, rare earth metal cryptate, europium tripyridyldiamine, bispyanin, La Jolla blue dye, allophycocyanin, allococcyanin B, phycocyanin C, phycocyanin R, phycoerythrin R, REG, rhodamine isothiocyanate, ROX, TAMRA, TET, TRIT (tetramethylrhodamine isothiol), tetramethylrhodamine, and Texas red.

4. The use according to claim 2, wherein the fluorescent substance further comprises rhodamine green or rhodamine red.

5. Use according to claim 1, wherein the indicator of signal intensity is selected from the group consisting of radioisotopes including¹¹⁰In、¹¹¹In、¹⁷⁷Lu、¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁹⁰Y、⁸⁹Zr、⁹⁴mTc、⁹⁴Tc、⁹⁹mTc、¹²⁰I、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、^154-158Gd、³²P、¹¹C、¹³N、¹⁵O、¹⁸⁶Re、¹⁸⁸Re、⁵¹Mn、⁵²mMn、⁵⁵Co、⁷²As、⁷⁵Br、⁷⁶Br、⁸²mRb and⁸³sr.

6. The use according to claim 2, wherein the enzyme comprises any one of horseradish peroxidase, alkaline phosphatase and glucose oxidase.

7. The use of any one of claims 1 to 6, wherein a test paper holder is arranged outside the test paper.

8. The use of claim 1, wherein the kit further comprises a risk indicator strip;

the risk indication strip is recorded with the relationship between the different signal intensities displayed by the detection area and the risk of lung cancer.

Images