CN111487415A - SNCG/NMP22 joint inspection colloidal gold test strip and preparation method and application thereof - Google Patents
SNCG/NMP22 joint inspection colloidal gold test strip and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides an SNCG/NMP22 joint inspection colloidal gold test strip, which sequentially comprises a sample pad, a gold conjugate pad, a reaction membrane and an absorption pad from a sample adding end, wherein the gold conjugate pad contains a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody, and the reaction membrane contains a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody. The SNCG/NMP22 joint-detection colloidal gold test strip prepared by the invention can complete routine detection in hospitals, can be carried to home to automatically complete detection, can improve the specificity and sensitivity of cancer detection compared with the colloidal gold test strip with a single antibody, and is more suitable for the common screening of tumors.
Description
Technical Field
The invention belongs to the field of tumor detection, and particularly relates to a gamma Synuclein (SNCG)/NMP22 colloidal gold test strip for detecting tumors, and a preparation method and application thereof.
Background
According to the data of the book 2015 for Chinese hygiene and family planning statistics, cancer becomes the first factor in the number of deaths caused by diseases in China. Particularly in the urban population, cancer causes death far more than heart disease in a proportion of 27%. Every day new cancer cases in the country exceed 1 million people, so cancer has become an important killer affecting the health of the national people. However, in the case of cancer incidence rates comparable to the world level in our country, mortality rates are higher than the world level (Chinese cancer mortality rates are higher than the global average 17%). The reason is that the characteristic high-incidence tumor in China is a digestive tract tumor with a low treatment level internationally, and the more important reason is that the cancer in China is mostly in a middle and late stage once found. Data from cancer, 2018, showed that 5-year survival rates for all cancers in the united states were 68% for white and 61% for black. The corresponding 5-year survival rate of Chinese tumor patients is only 36.9 percent.
The important factor that causes the death rate and the low survival rate of 5 years in China is that the early diagnosis of cancer is not fully paid attention. Cancers that are promising for cure are not diagnosed until the cancer is advanced, resulting in a compromised therapeutic outcome and premature death of the patient. Taking the detection of prostate cancer, which is a high-incidence cancer in american men, as an example, screening with Prostate Specific Antigen (PSA) in asymptomatic populations since the late 80 s directly led to the reduction of prostate cancer at a rate of more than 10% per year in 2010-2013. While the incidence of colorectal cancer is reduced by about 20% in adults 50 years and older in the united states due to the dramatic increase in the rate of colonoscope use (21% in 2000 to 60% in 2015). Therefore, the early diagnosis and early treatment of cancer have great significance for asymptomatic high-risk population and improving the survival rate of cancer patients, and bring great benefit for reducing the expense of government health and public utilities and reducing the burden of patients on families.
At present, most hospitals with more than two levels of hospitals have popularized large-scale imaging devices such as X-ray, ultrasound, L DCT, nuclear magnetism and the like, and liquid biopsy technologies developed by a plurality of companies for reducing the pain of patients caused by needle biopsy and invasive exploration are provided.
At present, the chemiluminescence reagent and the corollary equipment thereof mainly applied to large hospitals have the thresholds of high reagent and equipment cost and high later maintenance cost, and the medical cost of patients can be greatly increased. Therefore, the solution and the solution for detecting early cancer based on the detected protein tumor marker, which are flexible and portable, low in use cost, simple in training and operation, low-damage or even non-damage, simple and convenient in sampling and capable of realizing bedside detection, have the advantages that the development and the application of POCT are emphasized in the current market.
The gamma-prominent nucleoprotein (Synuclein-gamma, SNCG for short) is the third member of the Synuclein gene family identified by L avedan et al in 1998 from the brain genome library, is located at chromosome 10q 2312-q 2312, has a total length of about 510kb, contains 4 introns and 5 exons, and the high-expression SNCG can stimulate the proliferation, infiltration and metastasis of breast cancer cells.
The urinary nuclear matrix protein 22(NMP22) belongs to one of nuclear matrix proteins, and has been shown to be: the concentration of NMP22 in bladder cancer cells cultured in vitro was at least 25-fold higher than NMP22 in normal bladder epithelial cells. NMP22 is released after cell death and exists in human urine in the form of soluble compound or fragment, so NMP22 has become a protein tumor standard with high research value.
Disclosure of Invention
At present, a mature SNCG detection method does not exist, and the quantitative detection of NMP22 is based on an Elisa method, which has high sensitivity, but has complicated operation steps, is extremely time-consuming and is not beneficial to future clinical application.
Based on the defects of the prior art, the invention aims to provide an SNCG/NMP22 joint inspection colloidal gold test strip, a preparation method of the test strip and application of the test strip in preparation of a cancer detection kit.
The invention relates to an SNCG/NMP22 joint inspection colloidal gold test strip which is characterized in that,
a sample pad, a gold conjugate pad, a reaction membrane and an absorption pad are sequentially formed on the test strip from a sample adding end, wherein the gold conjugate pad contains a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody, and the reaction membrane contains a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody. In a particular aspect, the first murine anti-SNCG antibody of the test strip of the invention is an antibody recognizing gamma-prominent nuclear proteins and the second murine anti-SNCG antibody is an antibody recognizing gamma-prominent nuclear proteins different from the first murine anti-SNCG antibody.
In a specific aspect, the first anti-NMP 22 antibody of the test strip of the present invention is an antibody that recognizes the matrix protein 22 of the urinary nucleus, and the second anti-NMP 22 antibody is an antibody that recognizes the matrix protein 22 of the urinary nucleus that is different from the first anti-NMP 22 antibody.
In a specific aspect, the test strip of the invention has a first murine anti-SNCG antibody content of 0.1-1.5. mu.g/cm on the gold conjugate pad2Preferably 0.2 to 1.0. mu.g/cm2Most preferably 0.25 to 0.5. mu.g/cm2。
In a specific aspect, the test strip of the present invention has a first anti-NMP 22 antibody present in an amount of 0.1-1.5 μ g/cm on the gold conjugate pad2Preferably 0.15 to 1.0. mu.g/cm2Most preferably 0.17 to 0.33. mu.g/cm2。
In a specific aspect, the content of the second mouse anti-SNCG antibody on the reaction membrane of the test strip is 0.1-5.0 mu g/cm2Preferably 1.0 to 3.0. mu.g/cm2Most preferably 1.5 to 2.0. mu.g/cm2。
In a specific aspect, the content of the second anti-NMP 22 antibody on the reaction membrane of the test strip of the invention is 0.1-5.0 μ g/cm2Preferably 0.5 to 3.0μg/cm2Most preferably 1.0 to 2.0. mu.g/cm2。
The invention also relates to a preparation method of the SNCG/NMP22 joint inspection colloidal gold test strip, which comprises the following steps:
preparing colloidal gold particles;
combining the colloidal gold particles with a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody to form a colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate;
spraying and drying the mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate on a glass fiber membrane to obtain a gold conjugate pad;
preparing a detection line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane, wherein a solution containing a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody and a solution containing a goat anti-mouse IgG polyclonal antibody are respectively sprayed on the nitrocellulose membrane to form an SNCG detection line, an NMP22 detection line and a quality control line;
and (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
In a specific aspect, the first murine anti-SNCG antibody in the above preparation method is an antibody recognizing gamma-prominent nucleoprotein and the second murine anti-SNCG antibody is an antibody recognizing gamma-prominent nucleoprotein different from the first murine anti-SNCG antibody.
In a specific aspect, the first anti-NMP 22 antibody in the above preparation method is an antibody that recognizes the urine core matrix protein 22, and the second anti-NMP 22 antibody is an antibody that recognizes the urine core matrix protein 22 and is different from the first anti-NMP 22 antibody.
In a specific aspect, the colloidal gold particles are combined with the first mouse anti-SNCG antibody in the above preparation method, and the mass ratio of the colloidal gold particles to the first mouse anti-SNCG antibody is 5-40:1, preferably 8-30:1, and most preferably 10-20: 1.
In a specific aspect, the colloidal gold particles are combined with the first anti-NMP 22 antibody in the above preparation method, and the mass ratio of the colloidal gold particles to the first anti-NMP 22 antibody is 5-50:1, preferably 8-40:1, and most preferably 15-30: 1.
In a specific aspect, the above preparation method, in which the colloidal gold conjugate of the mouse anti-SNCG antibody/anti-NMP 22 antibody is sprayed on a glass fiber membrane, comprises re-dissolving the colloidal gold conjugate with a gold conjugate re-suspension, wherein the content of the first mouse anti-SNCG antibody after re-dissolving is 5-100 μ g/m L, preferably 10-50 μ g/m L, and most preferably 15-30 μ g/m L.
In a specific aspect, the above preparation method, in which the colloidal gold conjugate of the mouse anti-SNCG antibody/anti-NMP 22 antibody is sprayed on a glass fiber membrane, comprises re-dissolving the colloidal gold conjugate with a gold conjugate re-suspension, wherein the content of the first anti-NMP 22 antibody after re-dissolving is 5-100 μ g/m L, preferably 8-50 μ g/m L, and most preferably 10-20 μ g/m L.
In a specific aspect, the content of the second mouse anti-SNCG antibody in the solution containing the second mouse anti-SNCG antibody in the above preparation method is 0.5-5.0mg/m L, preferably 1.0-3.0mg/m L, most preferably 1.5-2.0mg/m L.
In a specific aspect, the content of the second anti-NMP 22 antibody in the solution containing the second anti-NMP 22 antibody in the above preparation method is 0.2 to 5.0mg/m L, preferably 0.5 to 3.0mg/m L, and most preferably 1.0 to 2.0mg/m L.
The invention also relates to a preparation method of the test strip, which comprises the following steps:
preparing colloidal gold particles;
combining the colloidal gold particles with a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody to form a colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate;
spraying and drying the mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate on a glass fiber membrane to obtain a gold conjugate pad;
preparing a detection line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane, wherein a solution containing a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody and a solution containing a goat anti-mouse IgG polyclonal antibody are respectively sprayed on the nitrocellulose membrane to form an SNCG detection line, an NMP22 detection line and a quality control line;
and (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
The invention also relates to application of the test strip in preparation of a disease detection kit, which is characterized in that the disease is one or more than two of bladder cancer, kidney cancer, prostate cancer and kidney stone.
In a particular aspect, the kit is for use in detecting a urine sample.
In a particular aspect, the disease detection kit utilizes the following steps to perform the detection:
dripping a certain amount of detection sample into a sample pad of the test strip;
after a period of time, the result is interpreted by naked eyes, or the test strip is inserted into the detector and the result is interpreted.
In a particular aspect, the criteria for visual interpretation are:
any position of the SNCG detection line and the NMP22 detection line shows red, the position of the quality control line shows red, and the result is positive;
the two positions of the SNCG detection line and the NMP22 detection line do not show red, the position of the quality control line shows red, and the result is negative;
the position of the quality control line does not display red, and the result is invalid.
In a specific aspect, the test strip is inserted into the detector, and the interpretation criteria are:
the SNCG concentration in the sample is more than 4mg/m L, and the result is positive;
the concentration of SNCG in the sample is less than or equal to 4mg/m L22 and is more than 10U/m L, and the result is positive;
the SNCG concentration in the sample is less than or equal to 4mg/m L22 concentration and less than or equal to 10U/m L, and the result is negative.
The invention also relates to a kit which comprises the test strip or the test strip prepared by the method.
In a specific aspect, the above kit is used for detecting diseases, wherein the diseases are selected from one or more than two of bladder cancer, kidney cancer, prostate cancer and kidney stone.
Specifically, the present invention relates to the following aspects:
the SNCG/NMP22 joint inspection colloidal gold test strip is characterized in that a sample pad, a gold conjugate pad, a reaction membrane and an absorption pad are sequentially formed on the test strip from a sample adding end, the gold conjugate pad contains a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody, and the reaction membrane contains a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody.
The test strip of claim 1, wherein the first murine anti-SNCG antibody is an antibody that recognizes gamma-prominent nucleoprotein and the second murine anti-SNCG antibody is an antibody that recognizes gamma-prominent nucleoprotein that is different from the first murine anti-SNCG antibody.
The test strip of claim 1 or 2, wherein the first anti-NMP 22 antibody is an antibody that recognizes the matrix protein 22 in the urine core, and the second anti-NMP 22 antibody is an antibody that recognizes the matrix protein 22 in the urine core, which is different from the first anti-NMP 22 antibody.
The test strip of any one of claims 1 to 3, wherein the content of the first murine anti-SNCG antibody on the gold conjugate pad is 0.1 to 1.5 μ g/cm2Preferably 0.2 to 1.0. mu.g/cm2Most preferably 0.25 to 0.5. mu.g/cm2。
The test strip of any one of claims 1 to 4, wherein the amount of the first anti-NMP 22 antibody on the gold conjugate pad is 0.1 to 1.5 μ g/cm2Preferably 0.15 to 1.0. mu.g/cm2Most preferably 0.17 to 0.33. mu.g/cm2。
The test strip of any one of items 1 to 5, wherein the content of the second mouse anti-SNCG antibody on the reaction membrane is 0.1 to 5.0 μ g/cm2Preferably 1.0 to 3.0. mu.g/cm2Most preferably 1.5 to 2.0. mu.g/cm2。
The test strip of any one of items 1 to 6, wherein the content of the second anti-NMP 22 antibody on the reaction membrane is 0.1 to 5.0 μ g/cm2Preferably 0.5 to 3.0. mu.g/cm2Most preferably 1.0 to 2.0. mu.g/cm2。
A preparation method of an SNCG/NMP22 joint inspection colloidal gold test strip is characterized by comprising the following steps:
preparing colloidal gold particles;
combining the colloidal gold particles with a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody to form a colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate;
spraying and drying the mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate on a glass fiber membrane to obtain a gold conjugate pad;
preparing a detection line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane, wherein a solution containing a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody and a solution containing a goat anti-mouse IgG polyclonal antibody are respectively sprayed on the nitrocellulose membrane to form an SNCG detection line, an NMP22 detection line and a quality control line;
and (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
The method of claim 8, wherein the first murine anti-SNCG antibody is an antibody that recognizes gamma-prominent nuclear protein and the second murine anti-SNCG antibody is a different antibody that recognizes gamma-prominent nuclear protein than the first murine anti-SNCG antibody.
The production method according to claim 8 or 9, wherein the first anti-NMP 22 antibody is an antibody that recognizes the urine core matrix protein 22, and the second anti-NMP 22 antibody is an antibody that recognizes the urine core matrix protein 22 and is different from the first anti-NMP 22 antibody.
The production method according to any one of items 8 to 10, wherein the colloidal gold particles are bound to the first mouse anti-SNCG antibody at a mass ratio of 5-40:1, preferably 8-30:1, and most preferably 10-20: 1.
The production method according to any one of items 8 to 11, wherein the colloidal gold particles are bound to the first anti-NMP 22 antibody at a mass ratio of 5 to 50:1, preferably 8 to 40:1, and most preferably 15 to 30:1 to the first anti-NMP 22 antibody.
The method according to any one of items 8 to 12, wherein the colloidal gold conjugate of the mouse anti-SNCG antibody/anti-NMP 22 antibody is sprayed on a glass fiber membrane, and the method comprises redissolving the colloidal gold conjugate with a gold conjugate resuspension, wherein the content of the first mouse anti-SNCG antibody after redissolving is 5-100 μ g/m L, preferably 10-50 μ g/m L, and most preferably 15-30 μ g/m L.
The method according to any one of items 8 to 13, wherein the colloidal gold conjugate of the mouse anti-SNCG antibody/anti-NMP 22 antibody is sprayed on a glass fiber membrane, and the method comprises redissolving the colloidal gold conjugate with a gold conjugate resuspension, wherein the content of the first anti-NMP 22 antibody after redissolving is 5 to 100 μ g/m L, preferably 8 to 50 μ g/m L, and most preferably 10 to 20 μ g/m L.
The method according to any one of items 8 to 14, wherein the content of the second mouse anti-SNCG antibody in the solution containing the second mouse anti-SNCG antibody is 0.5 to 5.0mg/m L, preferably 1.0 to 3.0mg/m L, and most preferably 1.5 to 2.0mg/m L.
The method according to any one of items 8 to 15, wherein the content of the second anti-NMP 22 antibody in the solution containing the second anti-NMP 22 antibody is 0.2 to 5.0mg/m L, preferably 0.5 to 3.0mg/m L, and most preferably 1.0 to 2.0mg/m L.
17 the method of making the test strip of any one of claims 1-7, comprising:
preparing colloidal gold particles;
combining the colloidal gold particles with a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody to form a colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate;
spraying and drying the mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate on a glass fiber membrane to obtain a gold conjugate pad;
preparing a detection line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane, wherein a solution containing a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody and a solution containing a goat anti-mouse IgG polyclonal antibody are respectively sprayed on the nitrocellulose membrane to form an SNCG detection line, an NMP22 detection line and a quality control line;
and (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
18, use of the test strip of any one of items 1 to 7 in preparation of a disease detection kit, wherein the disease is one or more selected from bladder cancer, kidney cancer, prostate cancer, and kidney stones.
The use according to claim 18, wherein the kit is for detecting a urine sample.
The use according to claim 18 or 19, wherein the disease detection kit is for detection using the following steps:
dripping a certain amount of detection sample into a sample pad of the test strip;
after a period of time, the result is interpreted by naked eyes, or the test strip is inserted into the detector and the result is interpreted.
Use according to item 20, wherein the criteria for visual interpretation are:
any position of the SNCG detection line and the NMP22 detection line shows red, the position of the quality control line shows red, and the result is positive;
the two positions of the SNCG detection line and the NMP22 detection line do not show red, the position of the quality control line shows red, and the result is negative;
the position of the quality control line does not display red, and the result is invalid.
The use of claim 20, wherein the test strip is inserted into the test device and the criteria for interpretation are:
the SNCG concentration in the sample is more than 4mg/m L, and the result is positive;
the concentration of SNCG in the sample is less than or equal to 4mg/m L22 and is more than 10U/m L, and the result is positive;
the SNCG concentration in the sample is less than or equal to 4mg/m L22 concentration and less than or equal to 10U/m L, and the result is negative.
A kit comprising the test strip of any one of items 1 to 7 or the test strip prepared by the method of any one of items 8 to 17.
The kit according to item 23, wherein the kit is used for detection of a disease selected from one or more of bladder cancer, kidney cancer, prostate cancer, and kidney stones.
The SNCG/NMP22 joint-detection colloidal gold test strip prepared by the invention can complete routine detection in hospitals, can be carried to home to automatically complete detection, can improve the specificity and sensitivity of cancer detection compared with the colloidal gold test strip with a single antibody, and is more suitable for the common screening of tumors.
Drawings
FIG. 1 is a structural diagram of the colloidal gold test strip of the present invention.
Description of reference numerals: 1-sample pad, 2-gold conjugate pad, 3-reaction membrane, 311-SNCG detection line, 312-NMP22 detection line, 32-quality control line and 4-absorption pad.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
As shown in FIG. 1, the SNCG/NMP22 joint inspection colloidal gold test strip of the present invention comprises a sample pad 1, a gold conjugate pad 2, a reaction membrane 3 and an absorption pad 4 formed in this order from the sample application end. Wherein, the reaction membrane 3 is provided with a detection line SNCG detection line 311, an NMP22 detection line 312 and a quality control line 32.
Wherein, the sample pad 1 is used for dripping a detection sample and is made of a glass cellulose membrane.
Specifically, the gold conjugate pad 2 is formed by spraying colloidal gold-labeled mouse anti-SNCG antibody colloidal gold conjugate on a glass cellulose membrane. Gold conjugate pad 2 contains a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody. The gold conjugate pad 2 may further contain bovine serum albumin and sodium caseinate.
Specifically, the material of the reaction membrane 3 is a nitrocellulose membrane. Wherein, a solution containing a second mouse anti-SNCG antibody is sprayed on a nitrocellulose membrane to form a detection line 311, a solution containing a second anti-NMP 22 antibody is sprayed on the nitrocellulose membrane to form a detection line 312, and a solution containing a goat anti-mouse IgG polyclonal antibody is sprayed on the nitrocellulose membrane to form a quality control line 32, wherein the positions of the detection line 311 and the detection line 312 can be interchanged.
As used herein, the first and second murine anti-SNCG antibodies are both antibodies that recognize gamma-prominent nucleoproteins, and the first and second murine anti-SNCG antibodies may be the same antibody or different antibodies, preferably, the first and second murine anti-SNCG antibodies are different. Those skilled in the art will appreciate that antibodies having such a function may be used, for example, the first murine anti-SNCG antibody may be secreted from a hybridoma cell line with a accession number of CGMCC No. 2469. The second mouse anti-SNCG antibody can be secreted by a hybridoma cell strain with the preservation number of CGMCC NO. 2468.
As used herein, the first anti-NMP 22 antibody and the second anti-NMP 22 antibody are both antibodies recognizing the urine nuclear matrix protein 22, and the first anti-NMP 22 antibody and the second anti-NMP 22 antibody may be the same antibody or different antibodies, and preferably, the first anti-NMP 22 antibody and the second anti-NMP 22 antibody are different. It will be appreciated by those skilled in the art that antibodies having such a function may be used, for example, the first anti-NMP 22 antibody is secreted by cell line 9F2 from Chongqing Producer science, Inc. The second anti-NMP 22 antibody was secreted by the cell line No. 6D7 of chongqing prosperous technologies ltd.
The invention also provides a preparation method of the SNCG/NMP22 joint inspection colloidal gold test strip, which comprises the following steps:
preparing colloidal gold particles;
combining the colloidal gold particles with a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody to form a colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate;
spraying and drying the mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate on a glass fiber membrane to obtain a gold conjugate pad;
preparing a detection line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane, wherein a solution containing a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody and a solution containing a goat anti-mouse IgG polyclonal antibody are respectively sprayed on the nitrocellulose membrane to form an SNCG detection line, an NMP22 detection line and a quality control line;
and (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
Specifically, the colloidal gold particles are combined with the first mouse anti-SNCG antibody, and the mass ratio of the colloidal gold particles to the first mouse anti-SNCG antibody is 5-40:1, preferably 8-30:1, and most preferably 10-20: 1. The mass ratio of the colloidal gold particles to the first anti-NMP 22 antibody is 5-50:1, preferably 8-40:1, and most preferably 15-30: 1.
Specifically, the colloidal gold conjugate of the mouse anti-SNCG antibody/anti-NMP 22 antibody is sprayed on a glass fiber membrane, and the colloidal gold conjugate is redissolved by using a gold conjugate resuspension solution, wherein the content of the first mouse anti-SNCG antibody after redissolution is 5-100 mu g/m L, preferably 10-50 mu g/m L, and most preferably 15-30 mu g/m L, and the content of the first anti-NMP 22 antibody after redissolution is 5-100 mu g/m L, preferably 8-50 mu g/m L, and most preferably 10-20 mu g/m L.
Specifically, the content of the second mouse anti-SNCG antibody in the solution containing the second mouse anti-SNCG antibody is 0.5-5.0mg/m L, preferably 1.0-3.0mg/m L, and most preferably 1.5-2.0mg/m L. the content of the second anti-NMP 22 antibody in the solution containing the second anti-NMP 22 antibody is 0.2-5.0mg/m L, preferably 0.5-3.0mg/m L, and most preferably 1.0-2.0mg/m L.
The invention also provides application of the test strip in preparation of a disease detection kit, wherein the disease is one or more than two of bladder cancer, kidney cancer, prostate cancer and kidney stone.
Wherein, the kit is used for detecting urine samples.
Specifically, the disease detection kit performs detection by using the following steps:
dripping a certain amount of detection sample into a sample pad of the test strip;
after a period of time, the result is interpreted by naked eyes, or the test strip is inserted into the detector and the result is interpreted.
The detector can be a quantitative detector or a semi-quantitative detector. For the semi-quantitative detector, the judgment criteria of the detection result are as follows:
the SNCG concentration in the sample is more than 4mg/m L, and the result is positive;
the concentration of SNCG in the sample is less than or equal to 4mg/m L22 and is more than 10U/m L, and the result is positive;
the SNCG concentration in the sample is less than or equal to 4mg/m L22 concentration and less than or equal to 10U/m L, and the result is negative.
For a quantitative detector, the numerical value of the detection result can be directly read.
In addition, the sample can also be directly dripped into the sample pad of the test strip, and then the detection result can be read by naked eyes. Wherein, the judgment standard of the detection result is as follows:
any position of the SNCG detection line and the NMP22 detection line shows red, the position of the quality control line shows red, and the result is positive;
the two positions of the SNCG detection line and the NMP22 detection line do not show red, the position of the quality control line shows red, and the result is negative;
the position of the quality control line does not display red, and the result is invalid.
The invention also relates to a kit which comprises the test strip or the test strip prepared by the method.
The kit is used for detecting diseases, wherein the diseases are selected from one or more than two of bladder cancer, kidney cancer, prostate cancer and kidney stone.
The test strip and the kit prepared by the invention can effectively detect bladder cancer, kidney cancer, prostate cancer and kidney stone, and have high sensitivity and specificity. The performance of detecting bladder cancer reaches more than 80%, the sensitivity of kidney cancer reaches more than 70%, the sensitivity of prostate cancer reaches more than 70%, the sensitivity of kidney stones reaches more than 60%, and the specificity in normal people reaches 93.3%.
Sensitivity in this context refers to the rate at which a positive test result is obtained from a sample of a patient with a well-defined clinical condition. Specificity in a normal human refers to the rate at which a test result is negative in a sample of a patient without a specific clinical disease.
Example 1
In the following examples, the materials used in the examples are all commercially available products unless otherwise specified.
(1) Preparation of colloidal gold particles
Weighing 1.0g of chloroauric acid, balancing to room temperature, weighing 2.0m L purified water, adding into a glass bottle filled with the chloroauric acid, repeatedly blowing and beating until the chloroauric acid is completely dissolved, pouring the dissolved chloroauric acid into a container, repeatedly washing the chloroauric acid glass bottle with the purified water for 5 times, pouring the washing liquid back into the container, fixing the volume of the solution to 10.0m L with the purified water, tightly covering the bottle cover, fully shaking for 15 minutes, uniformly mixing, and preparing into a 10% chloroauric acid solution.
Measuring a 10% chloroauric acid solution 0.3m L, adding the chloroauric acid solution into purified water 100.0m L, heating while stirring until boiling, measuring a 10% trisodium citrate solution 0.6m L, quickly and once adding the trisodium citrate solution into the boiled chloroauric acid solution, continuously heating and boiling for 2min, turning off a heat source, preheating and heating for 5min, cooling to room temperature, adding purified water to reach 100.0m L, and obtaining the solution containing the colloidal gold particles.
(2) Preparation of first and second murine anti-SNCG antibodies
The preparation method of the SNGG antibody described in Chinese patent application with the application number of 200810105770.7 and the invention name of the SNCG antibody and the application thereof is adopted, a hybridoma cell strain with the preservation number of CGMCC NO.2469 is used for secreting a first mouse anti-SNCG antibody, and a hybridoma cell strain with the preservation number of CGMCC NO.2468 is used for secreting a second mouse anti-SNCG antibody.
(3) Preparation of colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate
Adding 100.0M L (0.03g) of colloidal gold into a triangular flask, accurately adding 3.5M L0.1M of potassium carbonate solution, uniformly mixing, and standing for 10 minutes, adding 1.5mg of the first anti-SNCG antibody prepared in the step (2) and 1.8mg of the first anti-NMP 22 antibody (produced by a cell strain (Chongqing Tanshiki technology Co., Ltd.) numbered 9F 2) dropwise into the triangular flask under the condition of rapid stirring, standing for 40 minutes at room temperature, adding 10g/100M L of bovine serum albumin solution of 10.0M L dropwise into the triangular flask under the condition of rapid stirring, and standing for 15 minutes at room temperature to obtain the colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate.
(4) Preparation of gold conjugate pad
Centrifuging the above colloidal gold conjugate at 12000rpm at 4 deg.C for 40 min, carefully discarding the supernatant, adding the colloidal gold conjugate resuspension solution to original volume, redissolving the colloidal gold conjugate with the colloidal gold resuspension solution to obtain a redissolved solution containing 15 μ g/m L of the first mouse anti-SNCG antibody and 18 μ g/m L of the first anti-NMP 22 antibody, and spraying the redissolved solution on a glass fiber membrane with a spraying amount of 0.0166m L/cm2And dried to obtain a gold conjugate pad. Drying for 5 hr in a drying chamber after spraying to obtain gold conjugate pad, and storing in a room temperature drier.
Wherein, the colloidal gold conjugate resuspension is prepared as follows:
0.124g of boric acid, 0.762g of borax, 150.0g of cane sugar, 7.0g of bovine serum albumin, 1.0g of casein sodium, Triton X-1005.0 g, K305 g of polyvinylpyrrolidone, polyethylene glycol-200001 g and 700.0m L of purified water are added, stirred to be fully dissolved, a proper amount of concentrated hydrochloric acid is added, stirred uniformly, the pH value is adjusted to 7.4, and the volume is fixed to 1000m L after full stirring and dissolution.
(5) And preparing a detection line and a quality control line on the nitrocellulose membrane to obtain the reaction membrane.
Adding the second mouse anti-SNCG antibody and the second anti-NMP 22 antibody (produced by cell line No. 6D7 (Chongqing Prov. Tech., Ltd)) prepared in step (2) into PBS (5.0M L0.05.05M), mixing well, diluting with 0.05MPBS buffer solution to a final volume of 10.0M L to obtain a final concentration of 1.7mg/M L for the second mouse anti-SNCG and 1.0mg/M L for the second anti-NMP 22 antibody, and spraying the solution containing the second mouse anti-SNCG antibody and the solution containing the second anti-NMP 22 antibody to obtain a spraying amount of 0.1 mu L/cm2Spraying the solution on a nitrocellulose membrane to form an SNCG detection line and an NMP22 detection line respectively.
Adding 15.0mg of goat anti-mouse polyclonal antibody into 5.0M L0.05.05M PBS buffer solution, mixing well, diluting with 0.05MPBS buffer solution to a final volume of 10.0M L to make the final concentration of goat anti-mouse polyclonal antibody 1.5mg/M L, and mixing the solution containing goat anti-mouse IgG polyclonal antibody at 0.1 mu L/cm2Spraying the solution on a nitrocellulose membrane to form a quality control line. And (3) putting the reaction film of the coated detection line and the coated quality control line into a drying workshop for drying for 5 hours.
(6) And (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
The content of the first mouse anti-SNCG antibody on the gold conjugate pad on the prepared test strip is 0.25 mug/cm through calculation2The content of the first anti-NMP 22 antibody was 0.3. mu.g/cm2The content of the second mouse anti-SNCG antibody on the reaction membrane is 1.7 mu g/cm2The content of the second anti-NMP 22 antibody was 1.0. mu.g/cm2。
Example 2
(1) Preparation of colloidal gold particles
The preparation procedure was the same as in example 1.
(2) Preparation of first and second murine anti-SNCG antibodies
The preparation procedure was the same as in example 1.
(3) Preparation of colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate
The difference from example 1 is that 2.0mg of the primary mouse anti-SNCG antibody prepared in step (2) and 2.0mg of the primary anti-NMP 22 antibody were taken, and the 10g/100m L casein sodium solution of 1.5m L was changed to 10g/100m L casein sodium solution of 2.0m L, and other reaction conditions were the same as in example 1.
(4) Preparation of gold conjugate pad
The difference from example 1 was that the content of the first mouse anti-SNCG antibody after reconstitution was 20. mu.g/m L and the content of the first anti-NMP 22 antibody was 20. mu.g/m L, and other reaction conditions were the same as in example 1.
(5) And preparing a detection line and a quality control line on the nitrocellulose membrane to obtain the reaction membrane.
The difference from example 1 was that the final concentration of the second mouse anti-SNCG was 1.5mg/m L and the concentration of the second anti-NMP 22 antibody was 2.0mg/m L other reaction conditions were the same as example 1.
(6) The same procedure as in example 1.
The content of the first mouse anti-SNCG antibody on the gold conjugate pad on the prepared test strip is 0.33 mug/cm through calculation2The content of the first anti-NMP 22 antibody was 0.33. mu.g/cm2The content of the second mouse anti-SNCG antibody on the reaction membrane is 1.5 mu g/cm2The content of the second anti-NMP 22 antibody was 2.0. mu.g/cm2。
Example 3
(1) Preparation of colloidal gold particles
The preparation procedure was the same as in example 1.
(2) Preparation of first and second murine anti-SNCG antibodies
The preparation procedure was the same as in example 1.
(3) Preparation of colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate
The difference from example 1 is that 2.5mg of the primary mouse anti-SNCG antibody prepared in step (2) and 1.0mg of the primary anti-NMP 22 antibody were used and other reaction conditions were the same as in example 1.
(4) Preparation of gold conjugate pad
The difference from example 1 was that the content of the first mouse anti-SNCG antibody after reconstitution was made 25. mu.g/m L, the content of the first anti-NMP 22 antibody was made 10. mu.g/m L. Tween 20 was used in the preparation of colloidal gold conjugate resuspension, and other reaction conditions were the same as in example 1.
(5) And preparing a detection line and a quality control line on the nitrocellulose membrane to obtain the reaction membrane.
The difference from example 1 was that the final concentration of the second mouse anti-SNCG was 2.0mg/m L and the concentration of the second anti-NMP 22 antibody was 1.4mg/m L other reaction conditions were the same as example 1.
(6) The same procedure as in example 1.
The content of the first mouse anti-SNCG antibody on the gold conjugate pad on the prepared test strip is 0.42 mug/cm through calculation2The content of the first anti-NMP 22 antibody was 0.17. mu.g/cm2The content of the second mouse anti-SNCG antibody on the reaction membrane is 2.0 mu g/cm2The content of the second anti-NMP 22 antibody was 1.4. mu.g/cm2。
Example 4
(1) Preparation of colloidal gold particles
The preparation procedure was the same as in example 1.
(2) Preparation of first and second murine anti-SNCG antibodies
The preparation procedure was the same as in example 1.
(3) Preparation of colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate
The difference from example 1 is that 3.0mg of the primary mouse anti-SNCG antibody prepared in step (2) and 1.5mg of the primary anti-NMP 22 antibody were used and other reaction conditions were the same as in example 1.
(4) Preparation of gold conjugate pad
The difference from example 1 was that the content of the first mouse anti-SNCG antibody after reconstitution was 30. mu.g/m L and the content of the first anti-NMP 22 antibody was 15. mu.g/m L.
(5) And preparing a detection line and a quality control line on the nitrocellulose membrane to obtain the reaction membrane.
The difference from example 1 was that the final concentration of the second mouse anti-SNCG was 1.8mg/m L and the concentration of the second anti-NMP 22 antibody was 1.7mg/m L other reaction conditions were the same as example 1.
(6) The same procedure as in example 1.
Calculating to obtain test strip with the first mouse anti-SNCG antibody content of 0.5 μ g/cm on the gold conjugate pad2The content of the first anti-NMP 22 antibody was 0.25. mu.g/cm2The content of the second mouse anti-SNCG antibody on the reaction membrane is 1.8 mu g/cm2The content of the second anti-NMP 22 antibody was 1.7. mu.g/cm2。
Comparative example 1
Comparative example 1 is the detection of anti-NMP 22 antibody alone, not the combined detection of SNCG and NMP22 antibodies. The specific implementation steps are as follows:
(1) preparation of colloidal gold particles
The preparation procedure was the same as in example 1.
(2) Preparation of first and second murine anti-SNCG antibodies
The preparation procedure was the same as in example 1.
(3) Preparation of colloidal gold-labeled anti-NMP 22 antibody colloidal gold conjugate
The difference from example 1 was that only 2.0mg of the primary anti-NMP 22 antibody was taken, and the primary mouse anti-SNCG antibody was not added. Other reaction conditions were the same as in example 1.
(4) Preparation of gold conjugate pad
The difference from example 1 was that the content of the primary anti-NMP 22 antibody after reconstitution was made 20. mu.g/m L, and the primary mouse anti-SNCG antibody was not present other reaction conditions were the same as in example 1.
(5) And preparing a detection line and a quality control line on the nitrocellulose membrane to obtain the reaction membrane.
The difference from example 1 is that only NMP22 detection line was prepared so that the concentration of the second anti-NMP 22 antibody was 2.0mg/m L other reaction conditions were the same as example 1.
(6) The same procedure as in example 1.
Through calculation, the content of the first anti-NMP 22 antibody on the gold conjugate pad on the prepared test strip is 0.3 mu g/cm2The content of the second anti-NMP 22 antibody on the reaction membrane was 1.0. mu.g/cm2。
Specific reaction conditions for preparing the test strips in the above 4 examples and comparative examples are shown in table 1.
TABLE 1 reaction conditions for the preparation of test strips in the different examples
Example 6
The test paper prepared in the embodiment 1 is used for semi-quantitative detection of 4 diseases such as bladder cancer, kidney cancer, prostate cancer, kidney stone and the like.
Specifically, the test strip prepared in example 1 was inserted into a colloidal gold immunochromatographic assay apparatus (GD-010), and the urine of 120 μ L cancer (4 different diseases) patients and normal persons were respectively collected, wherein 15 normal persons, 10 bladder cancers, 10 kidney cancers, 10 prostate cancers, and 10 kidney stones were added (modified according to data results), and different urine was dropped onto the sample pad of the test strip, and the results were interpreted after 8 min.
Wherein the SNCG concentration in the sample is more than 4mg/m L, and the result is positive;
the concentration of SNCG in the sample is less than or equal to 4mg/m L22 and is more than 10U/m L, and the result is positive;
the SNCG concentration in the sample is less than or equal to 4mg/m L22 concentration and less than or equal to 10U/m L, and the result is negative.
The test results are shown in table 2. According to the results of the measurement in Table 2, the sensitivity of the kit for detecting bladder cancer was 80.0% (8/10), the sensitivity for kidney cancer was 70.0% (7/10), the sensitivity for prostate cancer was 70.0% (7/10), the sensitivity for kidney stones was 60.0% (6/10), and the specificity in normal humans was 93.3% (14/15).
TABLE 2.4 results of the test strip of example 1 for testing 4 kinds of disease and normal human samples
Example 7
The test paper prepared in the embodiment 2 is used for quantitative detection of 4 diseases such as bladder cancer, kidney cancer, prostate cancer, kidney stone and the like.
Specifically, the test strip prepared in example 2 was inserted into a colloidal gold immunochromatographic assay apparatus (GD-010), and urine of 100 μ L cancer (4 different diseases) patients and urine of normal persons were respectively taken, wherein 15 normal persons, 10 bladder cancers, 10 kidney cancers, 10 prostate cancers and 10 kidney stones were respectively added to the sample pad of the test strip, and the results were interpreted after 9 min.
Wherein the SNCG concentration in the sample is more than 4mg/m L, and the result is positive;
the concentration of SNCG in the sample is less than or equal to 4mg/m L22 and is more than 10U/m L, and the result is positive;
the SNCG concentration in the sample is less than or equal to 4mg/m L22 concentration and less than or equal to 10U/m L, and the result is negative.
The test results are shown in table 3. According to the results of the measurement in Table 3, the sensitivity of the present kit for detecting bladder cancer was 80.0% (8/10), kidney cancer was 70.0% (7/10), prostate cancer was 70.0% (7/10), kidney stones was 60.0% (6/10), and specificity in normal humans was 93.3% (14/15).
TABLE 3 test paper of example 2 results of 4 kinds of disease and normal human samples
Example 8
The test paper prepared in the embodiment 3 is used for macroscopic detection of 4 diseases such as bladder cancer, kidney cancer, prostate cancer, kidney stones and the like.
Specifically, the plasma of a 90 mu L cancer (4 different cancers) patient and the urine of a normal person are respectively taken, wherein 15 normal persons, 10 bladder cancers, 10 kidney cancers, 10 prostate cancers and 10 kidney stones are respectively added to a sample pad of a test strip, and the result is read after 10min of adding the different urine drops.
Any position of the SNCG detection line and the NMP22 detection line is red, the position of the quality control line is red, and the result is positive;
the two positions of the SNCG detection line and the NMP22 detection line do not show red, the position of the quality control line shows red, and the result is negative;
the position of the quality control line does not display red, and the result is invalid.
The test results are shown in table 4. According to the results of the measurement in Table 4, the sensitivity of the present kit for detecting bladder cancer was 80.0% (8/10), kidney cancer was 70.0% (7/10), prostate cancer was 70.0% (7/10), kidney stones was 60.0% (6/10), and specificity in normal humans was 93.3% (14/15).
TABLE 4 test paper of example 3 results of testing 4 kinds of disease and normal human samples
| Sample numbering | The result of the detection | Sample numbering | The result of the detection | Sample numbering | The result of the detection |
| Normal human-1 | Negative of | Normal human being 6 | Negative of | Normal human being 11 | Negative of |
| Normal human-2 | Negative of | Normal human-7 | Negative of | Normal human-12 | Negative of |
| Normal human-3 | Positive for | Normal human-8 | Negative of | Normal human-13 | Negative of |
| Normal human-4 | Negative of | Normal human-9 | Negative of | Normal human-14 | Negative of |
| Normal human-5 | Negative of | Normal human 10 | Negative of | Normal human-15 | Negative of |
| Bladder cancer-1 | Positive for | Kidney cancer-1 | Negative of | Prostate cancer-1 | Positive for |
| Bladder cancer-2 | Positive for | Kidney cancer-2 | Positive for | Prostate cancer-2 | Positive for |
| Bladder cancer-3 | Positive for | Kidney cancer-3 | Positive for | Prostate cancer-3 | Positive for |
| Bladder cancer-4 | Positive for | Kidney cancer-4 | Negative of | Prostate cancer-4 | Negative of |
| Bladder cancer-5 | Negative of | Kidney cancer-5 | Positive for | Prostate cancer-5 | Positive for |
| Bladder cancer-6 | Positive for | Kidney cancer-6 | Positive for | Prostate cancer-6 | Negative of |
| Bladder cancer-7 | Negative of | Kidney cancer-7 | Positive for | Prostate cancer-7 | Positive for |
| Bladder cancer-8 | Positive for | Kidney cancer-8 | Negative of | Prostate cancer-8 | Positive for |
| Bladder cancer-9 | Positive for | Kidney cancer-9 | Positive for | Prostate cancer-9 | Positive for |
| Bladder cancer-10 | Positive for | Kidney cancer-10 | Positive for | Prostate cancer-10 | Negative of |
| Kidney stone-1 | Positive for | Kidney stone-4 | Negative of | Kidney stone-7 | Positive for |
| Kidney stone-4 | Positive for | Kidney stone-5 | Positive for | Kidney stone-6 | Negative of |
| Kidney stone-7 | Positive for | Kidney stone-8 | Negative of | Kidney stone-9 | Positive for |
| Kidney stone-10 | Negative of | —— | —— | —— | —— |
Example 9
The test paper prepared in the embodiment 4 is used for semi-quantitative detection of 4 diseases such as bladder cancer, kidney cancer, prostate cancer, kidney stone and the like.
Specifically, the test strip prepared in example 4 was inserted into a colloidal gold immunochromatographic assay apparatus (GD-010), and urine was collected from 80 μ L cancer (4 different diseases) patients and normal persons, respectively, wherein 15 normal persons, 10 bladder cancers, 10 kidney cancers, 10 prostate cancers, and 10 kidney stones were added to the sample pad of the test strip, and the results were interpreted after 10 min.
Wherein the SNCG concentration in the sample is more than 4mg/m L, and the result is positive;
the concentration of SNCG in the sample is less than or equal to 4mg/m L22 and is more than 10U/m L, and the result is positive;
the SNCG concentration in the sample is less than or equal to 4mg/m L22 concentration and less than or equal to 10U/m L, and the result is negative.
The test results are shown in table 5. According to the results of the measurement in Table 5, the sensitivity of the present kit for detecting bladder cancer was 80.0% (8/10), kidney cancer was 70.0% (7/10), prostate cancer was 70.0% (7/10), kidney stones was 60.0% (6/10), and specificity in normal humans was 93.3% (14/15).
TABLE 5 test paper of example 4 results of 4 kinds of disease and normal human samples
| Sample numbering | The result of the detection | Sample numbering | The result of the detection | Sample numbering | The result of the detection |
| Normal human-1 | Negative of | Normal human being 6 | Negative of | Normal human being 11 | Negative of |
| Normal human-2 | Negative of | Normal human-7 | Negative of | Normal human-12 | Negative of |
| Normal human-3 | Positive for | Normal human-8 | Negative of | Normal human-13 | Negative of |
| Normal human-4 | Negative of | Normal human-9 | Negative of | Normal human-14 | Negative of |
| Normal human-5 | Negative of | Normal human 10 | Negative of | Normal human-15 | Negative of |
| Bladder cancer-1 | Positive for | Kidney cancer-1 | Negative of | Prostate cancer-1 | Positive for |
| Bladder cancer-2 | Positive for | Kidney cancer-2 | Positive for | Prostate cancer-2 | Positive for |
| Bladder cancer-3 | Positive for | Kidney cancer-3 | Positive for | Prostate cancer-3 | Positive for |
| Bladder cancer-4 | Positive for | Kidney cancer-4 | Negative of | Prostate cancer-4 | Negative of |
| Bladder cancer-5 | Negative of | Kidney cancer-5 | Positive for | Prostate cancer-5 | Positive for |
| Bladder cancer-6 | Positive for | Kidney cancer-6 | Positive for | Prostate cancer-6 | Negative of |
| Bladder cancer-7 | Negative of | Kidney cancer-7 | Positive for | Prostate cancer-7 | Positive for |
| Bladder cancer-8 | Positive for | Kidney cancer-8 | Negative of | Prostate cancer-8 | Positive for |
| Bladder cancer-9 | Positive for | Kidney cancer-9 | Positive for | Prostate cancer-9 | Positive for |
| Bladder cancer-10 | Positive for | Kidney cancer-10 | Positive for | Prostate cancer-10 | Negative of |
| Kidney stone-1 | Positive for | Kidney stone-4 | Negative of | Kidney stone-7 | Positive for |
| Kidney stone-4 | Positive for | Kidney stone-5 | Positive for | Kidney stone-6 | Negative of |
| Kidney stone-7 | Positive for | Kidney stone-8 | Negative of | Kidney stone-9 | Positive for |
| Kidney stone-10 | Negative of | —— | —— | —— | —— |
Example 10
The test strip prepared in the comparative example 1 is used for quantitative detection of 4 diseases such as bladder cancer, kidney cancer, prostate cancer, kidney stone and the like.
Specifically, the test strip prepared in comparative example 1 was inserted into a colloidal gold immunochromatographic assay apparatus (GD-010), and urine of 120 μ L cancer (4 different diseases) patients and normal persons were respectively collected, wherein 15 normal persons, 10 bladder cancers, 10 kidney cancers, 10 prostate cancers, and 10 kidney stones were respectively collected, and different urine was dropped onto the sample pad of the test strip, and the results were interpreted after 8 min.
Wherein, the NMP22 concentration in the sample is more than 10U/m L, and the result is positive;
the NMP22 concentration in the sample is less than or equal to 10U/m L, and the result is negative.
The test results are shown in table 6. According to the results of the measurement in Table 6, the sensitivity of the present kit for detecting bladder cancer was 60.0% (6/10), the sensitivity of kidney cancer was 50.0% (5/10), the sensitivity of prostate cancer was 60.0% (6/10), the sensitivity of kidney stones was 60.0% (6/10), and the specificity in normal humans was 93.3% (14/15).
TABLE 6 test paper of comparative example 1 results of 4 kinds of disease and normal human samples
| Sample numbering | The result of the detection | Sample numbering | The result of the detection | Sample numbering | The result of the detection |
| Normal human-1 | 3.2 | Normal human being 6 | 4.8 | Normal human being 11 | 1.9 |
| Normal human-2 | 3.2 | Normal human-7 | 8.6 | Normal human-12 | 3.8 |
| Normal human-3 | 12.8 | Normal human-8 | 1 | Normal human-13 | 7.7 |
| Normal human-4 | 4.8 | Normal human-9 | 2.9 | Normal human-14 | 2.9 |
| Normal human-5 | 22.1 | Normal human 10 | 22.1 | Normal human-15 | 16.3 |
| Bladder cancer-1 | 36.8 | Kidney cancer-1 | 8.9 | Prostate cancer-1 | 16.3 |
| Bladder cancer-2 | 9.8 | Kidney cancer-2 | 15.4 | Prostate cancer-2 | 40.3 |
| Bladder cancer-3 | 56 | Kidney cancer-3 | 17.3 | Prostate cancer-3 | 66.2 |
| Bladder cancer-4 | 86.4 | Kidney cancer-4 | 9.1 | Prostate cancer-4 | 1.9 |
| Bladder cancer-5 | 7.8 | Kidney cancer-5 | 6.7 | Prostate cancer-5 | 20.2 |
| Bladder cancer-6 | 20.8 | Kidney cancer-6 | 20.2 | Prostate cancer-6 | 8.6 |
| Bladder cancer-7 | 7.9 | Kidney cancer-7 | 15.4 | Prostate cancer-7 | 6.4 |
| Bladder cancer-8 | 33.6 | Kidney cancer-8 | 7.8 | Prostate cancer-8 | 47 |
| Bladder cancer-9 | 8.7 | Kidney cancer-9 | 6.7 | Prostate cancer-9 | 55.7 |
| Bladder cancer-10 | 33.6 | Kidney cancer-10 | 16.3 | Prostate cancer-10 | 4.5 |
| Kidney stone-1 | 17.7 | Kidney stone-2 | 23 | Kidney stone-3 | 10.6 |
| Kidney stone-4 | 6.7 | Kidney stone-5 | 11.5 | Kidney stone-6 | 7.9 |
| Kidney stone-7 | 30.7 | Kidney stone-8 | 8.6 | Kidney stone-9 | 19.6 |
| Kidney stone-10 | 9.7 |
Claims (10)
1. The SNCG/NMP22 joint inspection colloidal gold test strip is characterized in that a sample pad, a gold conjugate pad, a reaction membrane and an absorption pad are sequentially formed on the test strip from a sample adding end, the gold conjugate pad contains a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody, and the reaction membrane contains a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody.
2. The test strip of claim 1, wherein the first murine anti-SNCG antibody is an antibody that recognizes gamma-prominent nucleoprotein and the second murine anti-SNCG antibody is a different antibody that recognizes gamma-prominent nucleoprotein than the first murine anti-SNCG antibody.
3. The test strip of claim 1 or 2, wherein the first anti-NMP 22 antibody is an antibody that recognizes the matrix protein 22 in the urine core and the second anti-NMP 22 antibody is an antibody that recognizes the matrix protein 22 in the urine core that is different from the first anti-NMP 22 antibody.
4. The test strip of any one of claims 1 to 3, wherein the first murine anti-SNCG antibody is present in an amount of 0.1 to 1.5 μ g/cm on the gold conjugate pad2Preferably 0.2 to 1.0. mu.g/cm2Most preferably 0.25 to 0.5. mu.g/cm2。
5. The test strip of any one of claims 1 to 4, wherein the amount of the first anti-NMP 22 antibody on the gold conjugate pad is 0.1 to 1.5 μ g/cm2Preferably 0.15 to 1.0. mu.g/cm2Most preferably 0.17 to 0.33. mu.g/cm2。
6. The test strip of any one of claims 1 to 5, wherein the content of the second murine anti-SNCG antibody on the reaction membrane is 0.1-5.0 μ g/cm2Preferably 1.0 to 3.0. mu.g/cm2Most preferably 1.5 to 2.0. mu.g/cm2。
7. The test strip of any one of claims 1 to 6, wherein the content of the second anti-NMP 22 antibody on the reaction membrane is 0.1-5.0 μ g/cm2Preferably 0.5 to 3.0. mu.g/cm2Most preferably 1.0 to 2.0. mu.g/cm2。
8. A preparation method of an SNCG/NMP22 joint inspection colloidal gold test strip is characterized by comprising the following steps:
preparing colloidal gold particles;
combining the colloidal gold particles with a first mouse anti-SNCG antibody and a first anti-NMP 22 antibody to form a colloidal gold-labeled mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate;
spraying and drying the mouse anti-SNCG antibody/anti-NMP 22 antibody colloidal gold conjugate on a glass fiber membrane to obtain a gold conjugate pad;
preparing a detection line and a quality control line on a nitrocellulose membrane to obtain a reaction membrane, wherein a solution containing a second mouse anti-SNCG antibody and a second anti-NMP 22 antibody and a solution containing a goat anti-mouse IgG polyclonal antibody are respectively sprayed on the nitrocellulose membrane to form an SNCG detection line, an NMP22 detection line and a quality control line;
and (3) sticking the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad on a PVC rubber plate, sequentially arranging the sample pad, the gold conjugate pad, the reaction membrane and the absorption pad along the flow direction of the sample, and cutting to obtain the test strip.
9. The method of claim 8, wherein the first murine anti-SNCG antibody is an antibody that recognizes gamma-prominent nuclear protein and the second murine anti-SNCG antibody is a different antibody that recognizes gamma-prominent nuclear protein than the first murine anti-SNCG antibody.
10. The production method according to claim 8 or 9, wherein the first anti-NMP 22 antibody is an antibody that recognizes the urine core matrix protein 22, and the second anti-NMP 22 antibody is an antibody that recognizes the urine core matrix protein 22 and is different from the first anti-NMP 22 antibody.
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| CN101570575A (en) * | 2008-04-30 | 2009-11-04 | 北京市肿瘤防治研究所 | Sncg monoclonal antibody and application thereof |
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| CN101570575A (en) * | 2008-04-30 | 2009-11-04 | 北京市肿瘤防治研究所 | Sncg monoclonal antibody and application thereof |
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| CN105899953A (en) * | 2013-11-05 | 2016-08-24 | 新加坡科技研究局 | Bladder carcinoma biomarkers |
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