CN112946291A

CN112946291A - Application of FGF18 in preparation of reagent for diagnosing and predicting ovarian cancer and FGF18 chemiluminescence detection kit

Info

Publication number: CN112946291A
Application number: CN201911256175.8A
Authority: CN
Inventors: 庄光磊; 狄文; 殷霞; 师凯旋
Original assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Renji Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2021-06-11

Abstract

The invention relates to the technical field of medical diagnosis, in particular to application of FGF18 in preparation of a reagent for diagnosing and predicting ovarian cancer and an FGF18 chemiluminescence detection kit. The invention has the advantages that: the FGF18 protein is found to be used as a novel ovarian cancer diagnosis marker for the first time, and has a 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

Application of FGF18 in preparation of reagent for diagnosing and predicting ovarian cancer and FGF18 chemiluminescence detection kit

Technical Field

The invention relates to the technical field of medical diagnosis, in particular to application of FGF18 (fibroblast growth factor 18) in preparation of a reagent or a kit for diagnosing and predicting ovarian cancer, and also relates to a FGF18 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, no report is known about the application of FGF18 in the preparation of a reagent or a kit for diagnosing and predicting ovarian cancer.

Disclosure of Invention

The invention aims to provide application of FGF18 protein in preparation of a reagent or a kit for diagnosing and predicting ovarian cancer, explain the significance of a novel protein marker FGF18 in diagnosis and prediction of ovarian cancer, and develop a detection kit of the protein marker FGF18 by adopting a chemiluminescence method.

In order to achieve the purpose, the invention adopts the technical scheme that: the application of FGF18 in preparing a reagent or a kit for diagnosing and predicting ovarian cancer is explained, and the corresponding FGF18 magnetic particle chemiluminescence detection kit is prepared accordingly.

1. The raw material for FGF18 detection is obtained by adopting a mode of self-expressing FGF18 protein and preparing an FGF18 antibody.

2. And coupling the FGF18 capture antibody to the magnetic beads by adopting a mode that proteins are coupled to the magnetic beads to obtain the FGF18 capture carrier. And (3) labeling alkaline phosphatase onto the detection antibody of the FGF18 by using a corresponding labeling method to obtain the FGF18 detection antibody. And (3) diluting the FGF18 standard protein by corresponding gradient to obtain a detection standard of FGF 18.

3. The method comprises the steps of adopting corresponding number of normal human and ovarian cancer patient serums as FGF18 detection samples, uniformly mixing and reacting FGF18 capture carriers with a certain amount of samples, washing the samples with cleaning solution, adding FGF18 detection antibodies to be combined with FGF18 proteins of the FGF18 capture carriers, then washing out foreign proteins and unbound detection antibodies, detecting reaction substrates, reading detection signals on a detection instrument, corresponding signal values to standard curves to obtain corresponding values, and comparing FGF18 content difference between normal human and ovarian cancer patients.

In a first aspect of the invention, the use of FGF18 in the preparation of a diagnostic marker for ovarian cancer is provided.

In a second aspect of the invention, there is provided the use of FGF18 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 FGF18 magnetic particle chemiluminescence detection reagent or kit.

Furthermore, the diagnostic reagent or the kit is a reagent or a kit for detecting the expression level of FGF18 in plasma.

Furthermore, the diagnostic reagent or the kit is a diagnostic reagent or a kit for detecting the expression level of FGF18 in blood plasma by using FGF18 as a biomarker and using a magnetic particle chemiluminescence detection kit.

Further, when the magnetic particle chemiluminescence detection kit detects that the concentration of FGF18 in plasma is higher than 150ng/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 FGF 18. The antibody may be a monoclonal antibody or a polyclonal antibody.

Furthermore, the diagnostic kit comprises FGF18 protein, FGF18 coupled magnetic beads, an AP labeled FGF18 detection antibody, a sample diluent, a washing buffer and a chemiluminescent substrate.

In a third aspect of the invention, a kit for diagnosing and predicting ovarian cancer is provided, wherein the kit is used for detecting the expression level of FGF18 in plasma.

Furthermore, the kit for diagnosing and predicting ovarian cancer comprises FGF18 protein, FGF18 coupled magnetic beads, AP labeled FGF18 detection antibody, sample diluent, washing buffer and chemiluminescent substrate.

The invention has the advantages that:

1. the FGF18 protein is found to be used as a novel 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 FGF18 chemiluminescent detection kit.

FIG. 2 is a scatter plot of FGF18 content for ovarian cancer patients and healthy people.

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 FGF18, using magnetic particle chemiluminescence.

FGF18 coupled magnetic bead preparation: 1mg of FGF18 antibody was diluted to 1mg/ml with PBS, 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 FGF18 detection antibody: the marker is alkaline phosphatase AP, and the FGF18 antibody is labeled by using an alkaline phosphatase Labeling kit-NH2 according to the instruction.

FGF18 protein standards were diluted to 1000, 500, 250, 125, 62.5, 31.25, 15.625, 0ng/ml, eight concentration gradients.

The experimental steps are as follows:

1. taking 100 ul of the protein sample, resuspending the protein sample with 50 ul of FGF18 coupled magnetic beads, and carrying out 15min at 37 ℃;

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 μ l of AP-labeled FGF18 detection antibody, resuspending, and performing at 37 deg.C 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: FGF18 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 FGF18 in the plasma of ovarian cancer patients is 251ng/ml, the mean content value of FGF18 in the plasma of normal people is 132ng/ml, the increase of patients is 90% compared with normal people, and the significant difference is significant, so that the FGF18 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

Use of FGF18 in the preparation of a diagnostic marker for ovarian cancer.
Use of FGF18 in the preparation of a reagent or kit for the diagnosis and prognosis of ovarian cancer.
3. The use of FGF18 in the preparation of a reagent or a kit for diagnosing and prognosing ovarian cancer according to claim 2, wherein the reagent or the kit for diagnosing and prognosing ovarian cancer is a reagent or a kit for magnetic particle chemiluminescence detection of FGF 18.
4. The use of FGF18 as claimed in claim 2, in the preparation of a reagent or kit for diagnosing and prognosing ovarian cancer, wherein the diagnostic reagent or kit is a reagent or kit for detecting the expression level of FGF18 in plasma.
5. The use of FGF18 in the preparation of a reagent or a kit for diagnosing and prognosing ovarian cancer according to claim 2, wherein the reagent or the kit is a reagent or a kit for diagnosing the expression level of FGF18 in plasma by using FGF18 as a biomarker and using a magnetic particle chemiluminescence detection kit.
6. The use of FGF18 of claim 5 in the preparation of a reagent or kit for diagnosing and prognosing ovarian cancer, wherein a magnetic particle chemiluminescence detection kit detects a concentration of FGF18 in plasma of greater than 150pg/ml, indicating that the patient is likely to have ovarian cancer.
7. The use of FGF18 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 magnetic particle chemiluminescence assay kit prepared using antibodies to FGF 18.
8. The use of FGF18 in the preparation of a reagent or kit for the diagnosis and prognosis of ovarian cancer according to claim 2, wherein the kit comprises FGF18 protein, FGF 18-coupled magnetic beads, AP-labeled FGF18 detection antibody, sample diluent, wash buffer, and a chemiluminescent substrate.
9. A kit for diagnosing and predicting ovarian cancer, which is used for detecting the expression level of FGF18 in plasma.
10. The kit of claim 9, wherein the kit comprises FGF18 protein, FGF 18-conjugated magnetic beads, AP-labeled FGF18 detection antibody, sample diluent, wash buffer, and chemiluminescent substrate.