CN112946290A - Application of extracellular matrix substrate response protein 1 in preparation of reagent for diagnosing and predicting ovarian cancer - Google Patents
Application of extracellular matrix substrate response protein 1 in preparation of reagent for diagnosing and predicting ovarian cancer Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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Abstract
The invention relates to the technical field of medical diagnosis, in particular to application of extracellular matrix substrate reaction protein 1 in preparation of a reagent for diagnosing and predicting ovarian cancer and a chemiluminescence detection kit for the extracellular matrix substrate reaction protein 1. The invention has the advantages that: the invention discovers that the extracellular matrix substrate response protein 1 protein is used as a new ovarian cancer diagnosis marker for the first time, and has key significance for diagnosis and prediction of ovarian cancer; the invention adopts the magnetic particle chemiluminescence method to develop the kit, is favorable for improving the detection sensitivity and the operation convenience compared with the enzyme-linked immunosorbent assay technology, and is also favorable for developing detection products matched with an automatic detection instrument in the later period.
Description
Technical Field
The invention relates to the technical field of medical diagnosis, in particular to application of Spondin1 (extracellular matrix substrate reaction protein 1, Spondin 1extracellular matrix protein) in preparation of a reagent or a kit for diagnosing and predicting ovarian cancer, and also relates to a Spondin1 chemiluminescence detection kit.
Background
CA125 is one of the common indicators for diagnosing ovarian cancer and detecting the recurrence of ovarian cancer, but CA125 is often not detected in early onset of patients, and the clinical application of CA125 has certain limitations. In view of this, researchers would like to find a more effective marker for the diagnosis and prognosis of ovarian cancer.
The traditional protein marker detection technology is still an enzyme-linked immunosorbent assay technology and has the characteristics of complex operation, unstable signals, inaccurate result judgment and the like.
Chinese patent document CN102735846A discloses a chemiluminescent immunoassay kit for ovarian cancer tumor marker HE4, and also discloses a method for detecting ovarian cancer tumor marker HE4 by using the chemiluminescent immunoassay kit for ovarian cancer tumor marker HE 4. The kit for detecting ovarian cancer tumor marker HE4 by chemiluminescence immunoassay comprises a microporous plate coated by 9F3 antibody, 10E1 antibody marked by alkaline phosphatase, analysis buffer solution, substrate working solution, luminescent substrate, washing solution and quality control product. The ovarian cancer tumor marker HE4 chemiluminescence immunoassay kit provided by the invention has a linear range of 0.5 ng/ml-1200 ng/ml and a detection limit of 0.2 ng/ml, and can be used for clinical auxiliary diagnosis, curative effect observation and prognosis judgment of ovarian cancer. However, there is no report on the application of Spondin1 in the preparation of reagent or kit for diagnosing and predicting ovarian cancer.
Disclosure of Invention
The invention aims to provide application of Spondin1 protein in preparation of a reagent or a kit for diagnosing and predicting ovarian cancer, explains the significance of a novel protein marker Spondin1 in diagnosis and prediction of ovarian cancer, and develops a detection kit of the protein marker Spondin1 by adopting a chemiluminescence method.
In order to achieve the purpose, the invention adopts the technical scheme that: the application of Spondin1 in preparing a reagent or a kit for diagnosing and predicting ovarian cancer is explained, and the corresponding Spondin1 magnetic particle chemiluminescence detection kit is prepared accordingly.
1. The method adopts the mode of self-expressing the Spondin1 protein and preparing the Spondin1 antibody to obtain the raw material for detecting the Spondin 1.
2. Coupling the Spondin1 capture antibody to the magnetic beads in a mode of coupling protein to the magnetic beads to obtain the Spondin1 capture carrier. And (3) labeling alkaline phosphatase onto the detection antibody of the Spondin1 by adopting a corresponding labeling method to obtain the Spondin 18 detection antibody. And (3) carrying out corresponding gradient dilution on the Spondin1 standard substance protein to obtain a detection standard substance of Spondin 1.
3. The method comprises the steps of taking corresponding number of serums of normal people and ovarian cancer patients as detection samples of Spondin1, uniformly mixing Spondin1 capture carriers with a certain amount of samples for reaction, washing the samples with cleaning solution, adding Spondin1 detection antibodies to be combined with Spondin1 proteins of the Spondin1 capture carriers, washing out foreign proteins and unbound detection antibodies, detecting reaction substrates, reading detection signals on a detection instrument, enabling signal values to correspond to standard curves to obtain corresponding values, and comparing Spondin1 content difference between the normal people and the ovarian cancer patients.
In a first aspect of the invention, the application of Spondin1 in preparing ovarian cancer diagnosis markers is provided.
In a second aspect of the invention, there is provided the use of Spondin1 in the preparation of a reagent or kit for the diagnosis and prognosis of ovarian cancer.
Furthermore, the reagent or the kit for diagnosing and predicting ovarian cancer is a reagent or a kit for detecting Spondin1 magnetic particle chemiluminescence method.
Furthermore, the diagnostic reagent or the kit is a reagent or a kit for detecting the expression level of the Spondin1 in the blood plasma.
Furthermore, the diagnostic reagent or the kit is used for detecting the expression level of the Spondin1 in the blood plasma by using the magnetic particle chemiluminescence detection kit with the Spondin1 as a biomarker.
Further, when the magnetic particle chemiluminescence detection kit detects that the concentration of Spondin1 in plasma is higher than 2670pg/ml, the patient is suggested to be likely to have ovarian cancer.
Furthermore, the diagnostic reagent or the kit is a magnetic particle chemiluminescence detection kit prepared by using an antibody of Spondin 1. The antibody may be a monoclonal antibody or a polyclonal antibody.
Furthermore, the diagnostic kit comprises Spondin1 protein, Spondin1 coupled magnetic beads, AP labeled Spondin1 detection antibody, sample diluent, washing buffer and chemiluminescent substrate.
In a third aspect of the invention, a kit for diagnosing and predicting ovarian cancer is provided, wherein the kit is a kit for detecting the expression level of Spondin1 in plasma.
Furthermore, the kit for diagnosing and predicting ovarian cancer comprises Spondin1 protein, Spondin1 coupled magnetic beads, AP labeled Spondin1 detection antibody, sample diluent, washing buffer and chemiluminescent substrate.
The invention has the advantages that:
1. the invention discovers that the Spondin1 protein is used as a new ovarian cancer diagnosis marker for the first time, and has a key significance for diagnosis and prediction of ovarian cancer;
2. the invention adopts the magnetic particle chemiluminescence method to develop the kit, is favorable for improving the detection sensitivity and the operation convenience compared with the enzyme-linked immunosorbent assay technology, and is also favorable for developing detection products matched with an automatic detection instrument in the later period.
Drawings
FIG. 1 is a standard curve of the Spondin1 chemiluminescence detection kit.
FIG. 2 is a scatter plot of Spondin1 content for ovarian cancer patients and healthy populations.
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1:
this example establishes a double antibody sandwich system of Spondin1, using magnetic particle chemiluminescence.
Preparation of Spondin1 coupled magnetic beads: 1mg of Spondin1 antibody was diluted to 1mg/ml with PBS (labeling buffer), 2mg of Biotin Sulfo-NHS-Biotin was dissolved in 100. mu.l of ultrapure water, and the two solutions were mixed and purified by passing through an ultrafiltration tube at room temperature for 30min or on ice for 2 hours. And (3) taking the purified solution, diluting to 0.5mg/ml, wherein the volume is 200 mu l, mixing the purified solution with 500 mu l of activated streptavidin magnetic beads, reacting, shaking for 10-30min at room temperature, washing for three times by using a washing solution, and resuspending and storing by using a magnetic bead preservation solution.
Labeling of Spondin1 detection antibody: the marker is alkaline phosphatase AP, and the reagent kit is labeled by using alkaline phosphatase Labeling kit-NH2, and the Spondin1 antibody is labeled according to the instruction.
The Spondin1 protein standard was diluted to 8000, 4000, 2000, 1000, 500, 250, 125, 0pg/ml, eight concentration gradients.
The experimental steps are as follows:
1. taking 100 ul of the protein sample, and resuspending the protein sample with 50 ul of Spondin1 coupled magnetic beads at 37 ℃ for 15 min;
2. adsorbing magnetic particles on a magnetic frame for 15s, adding 500 mu l of cleaning solution, cleaning, adsorbing the magnetic particles on the magnetic frame for 15s, and cleaning for three times;
3. adding 100 mul of detection antibody of the AP labeled Spondin1 for resuspension, 37 ℃ for 15 min;
4. adsorbing magnetic particles on a magnetic frame for 15s, adding 500 mu l of cleaning solution, cleaning, adsorbing the magnetic particles on the magnetic frame for 15s, and cleaning for three times;
5. a luminescent substrate (100. mu.l) was added to carry out signal detection.
The standard is shown in figure 1, and the result shows that: spondin1 double-antibody sandwich detection effect is good, the existing standard curve is adopted for detection, and R of the standard curve is2>0.99, the correlation is good, and the method can be used for the next detection.
Example 2:
20 ovarian cancer samples and 19 normal human samples were tested using the test system of example 1.
100 μ l of plasma samples were taken and tested using the system of example 1, and the gradient values of the standards were the gradients of example 1, relative to the standard, and the experimental procedure was as in example 1.
The concentration values of the results are shown in Table 1:
TABLE 1
As shown in figure 2, the mean content value of Spondin1 in the blood plasma of ovarian cancer patients is 3114pg/ml, while the mean content value of Spondin1 in the blood plasma of normal people is 2277pg/ml, and the increase of the patients is 37% compared with normal people, so that the significant difference is realized, and the Spondin1 serving as a marker for detecting ovarian cancer has the functions of diagnosing and distinguishing ovarian cancer patients from normal people, and has important significance for the production of later-stage reagents.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
Claims (10)
- Application of Spondin1 in preparation of ovarian cancer diagnostic markers.
- Application of Spondin1 in preparation of reagent or kit for diagnosing and predicting ovarian cancer.
- 3. The use of Spondin1 in the preparation of a reagent or kit for diagnosing and prognosing ovarian cancer according to claim 2, wherein the reagent or kit for diagnosing and prognosing ovarian cancer is a Spondin1 magnetic particle chemiluminescence assay reagent or kit.
- 4. The use of Spondin1 in the preparation of a reagent or kit for diagnosing and prognosing ovarian cancer according to claim 2, wherein the diagnostic reagent or kit is a reagent or kit for detecting the expression level of Spondin1 in plasma.
- 5. The use of Spondin1 in the preparation of a reagent or kit for diagnosing and prognosing ovarian cancer according to claim 2, wherein the reagent or kit is a diagnostic reagent or kit for detecting the expression level of Spondin1 in plasma by using Spondin1 as a biomarker and using a magnetic particle chemiluminescence detection kit.
- 6. Use of Spondin1 in the preparation of a reagent or kit for diagnosing and prognosing ovarian cancer according to claim 5, wherein when the magnetic particle chemiluminescence detection kit detects a concentration of Spondin1 in the plasma of more than 2670pg/ml, the patient is indicated as likely to have ovarian cancer.
- 7. The use of Spondin1 in the manufacture of a reagent or kit for the diagnosis and prognosis of ovarian cancer as claimed in claim 2, wherein the diagnostic reagent or kit is a magnetic particle chemiluminescence assay kit prepared using antibodies to Spondin 1.
- 8. The use of Spondin1 in the preparation of a reagent or kit for the diagnosis and prognosis of ovarian cancer according to claim 2, wherein the kit comprises Spondin1 protein, Spondin1 coupled magnetic beads, AP-labeled Spondin1 detection antibody, sample diluent, wash buffer and chemiluminescent substrate.
- 9. A kit for diagnosing and predicting ovarian cancer is characterized in that the kit is a kit for detecting the expression level of Spondin1 in plasma.
- 10. The kit of claim 9, wherein the kit comprises a Spondin1 protein, Spondin1 coupled magnetic beads, AP-labeled Spondin1 detection antibody, sample diluent, wash buffer, and chemiluminescent substrate.
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1964986A (en) * | 2003-10-07 | 2007-05-16 | 千年药品公司 | Nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer |
US20090087849A1 (en) * | 2007-09-06 | 2009-04-02 | Tripath Imaging, Inc. | Nucleic acid-based methods and compositions for the detection of ovarian cancer |
CN101410715A (en) * | 2006-01-27 | 2009-04-15 | 三路影像公司 | Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor |
US20100227335A1 (en) * | 2009-03-05 | 2010-09-09 | Becton, Dickinson And Company | Matrix metalloproteinase-7 (mmp-7) monoclonal antibodies and methods for their use in the detection of ovarian cancer |
CN102735846A (en) * | 2012-06-15 | 2012-10-17 | 河南生生医疗器械有限公司 | Chemiluminescence immunodetection kit and detection method for ovarian cancer tumor marker HE4 |
CN103389381A (en) * | 2013-07-19 | 2013-11-13 | 武汉生之源生物科技有限公司 | Human epididymal secretory protein E4 chemiluminescence detection kit and preparation method thereof |
-
2019
- 2019-12-10 CN CN201911256160.1A patent/CN112946290A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1964986A (en) * | 2003-10-07 | 2007-05-16 | 千年药品公司 | Nucleic acid molecules and proteins for the identification, assessment, prevention, and therapy of ovarian cancer |
CN101410715A (en) * | 2006-01-27 | 2009-04-15 | 三路影像公司 | Methods for identifying patients with an increased likelihood of having ovarian cancer and compositions therefor |
US20090087849A1 (en) * | 2007-09-06 | 2009-04-02 | Tripath Imaging, Inc. | Nucleic acid-based methods and compositions for the detection of ovarian cancer |
US20100227335A1 (en) * | 2009-03-05 | 2010-09-09 | Becton, Dickinson And Company | Matrix metalloproteinase-7 (mmp-7) monoclonal antibodies and methods for their use in the detection of ovarian cancer |
CN102735846A (en) * | 2012-06-15 | 2012-10-17 | 河南生生医疗器械有限公司 | Chemiluminescence immunodetection kit and detection method for ovarian cancer tumor marker HE4 |
CN103389381A (en) * | 2013-07-19 | 2013-11-13 | 武汉生之源生物科技有限公司 | Human epididymal secretory protein E4 chemiluminescence detection kit and preparation method thereof |
Non-Patent Citations (3)
Title |
---|
PYLE-CHENAULT RA 等: "VSGP/F-spondin: a new ovarian cancer marker", TUMOUR BIOL, vol. 26, no. 5, pages 245 - 257 * |
TING-TING JIAO 等: "Importance of spondin 1 and cellular retinoic acid binding protein 1 in the clinical diagnosis of ovarian cancer", J CLIN EXP PATHOL, vol. 6, no. 12, pages 3036 * |
张立会;王宏宇;刘红燕;聂建国;: "卵巢浆液性囊腺癌与交界性囊腺瘤的差异蛋白质组学分析", 中国实验诊断学, no. 05, pages 872 - 876 * |
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