CN111521807B - Spondin1 and CA125 combined used as early ovarian cancer biomarker and kit - Google Patents

Abstract

The invention relates to a biomarker and a kit for early ovarian cancer by combining Spondin1 and CA125, belonging to the technical field of immunodetection. The invention discloses application of a reagent for detecting the levels of Spondin1 and CA125 in a sample from a subject in preparing a kit for predicting, evaluating or diagnosing early ovarian cancer of the subject. The kit provided by the invention can be used for obviously improving the sensitivity and specificity of the prediction, evaluation and diagnosis of ovarian cancer in a subject and greatly reducing the false positive rate in the detection of early ovarian cancer.

Description

Spondin1 and CA125 combined used as early ovarian cancer biomarker and kit

Technical Field

The invention relates to the technical field of immunodetection in general, and particularly relates to a combination of Spondin1 and CA125 as an early ovarian cancer biomarker and a kit prepared from the early ovarian cancer biomarker.

Background

Ovarian cancer is one of the common malignant tumors of female reproductive organs, and poses a serious threat to female life. Because the ovary is deeply located in the pelvic cavity, the size is small, and typical symptoms are lacked during the onset of the ovarian cancer, the diagnosis of early ovarian cancer is difficult, and the ovarian cancer often develops to the middle and late stages during clinical discovery. And progresses to the middle and advanced stages, particularly after metastasis occurs, and the recurrence rate and the 5-year survival rate are low even if the ovarian epithelial cancer is treated by surgery. Therefore, early diagnosis of ovarian cancer is of great significance to reduce metastasis and improve 5-year survival rate. Currently, in clinic, the diagnosis of ovarian cancer is mainly based on vaginal ultrasonography (TVU) and detection of the level of carbohydrate antigen 125 (CA 125) in blood. However, the former requires a very high skill and experience from the doctor, has a low detectable rate, and is difficult to determine whether a tumor is benign or malignant, and therefore, it is necessary to determine the tumor by combining the detection results of tumor markers in blood.

CA125 (carbohydrate antigen) 125, also known as mucin-16, isMUC16A protein encoded by the gene. CA125 is one of the markers of ovarian epithelial cancer, and is widely used for diagnosis and follow-up of ovarian epithelial cancer at present. However, CA125 is also increased to varying degrees in the case of certain non-malignant diseases, and thus, when CA125 is used to diagnose ovarian cancer, neither specificity nor sensitivity is desirable. In recent years, joint detection using epididymisprotein 4 (epididymisprotein 4) and CA125 has been recommended for screening and diagnosis of ovarian cancer (patent documents 1 and 2). However, the sensitivity of single HE4 to the auxiliary diagnosis of stage I ovarian cancer is higher than that of CA125, but is only 45.9%; the sensitivity of The combined detection of HE4 and CA125 was not improved in stage I ovarian cancer (see Moore RG, Brown AK, Miller MC et al, The use of multiple novel tumor biomakers for The detection of ovarian cancer with a viral mass.Gynecol Oncol, 2008,108:402-408). Therefore, there is an urgent need to develop new serum markers for early stage ovarian cancer with diagnostic value, so as to determine the occurrence of ovarian cancer by peripheral blood detection sensitively and specifically in the early stage of ovarian cancer.

Spondin1 protein (accession number UniProtKB-Q9HCB6 in uniprot. org), also known as F-Spondin, consisting ofSPON1The gene code, 807 amino acids in length, is a cell adhesion protein that promotes the attachment of spinal cord and sensory neuron cells and the growth of neurites in vitro. By similarity analysis, it is thought that it may contribute to the growth and guidance of axons in the spinal cord and peripheral nervous system. However, the combination of CA125 and Spondin1 for biomarkers for early ovarian cancer diagnosis has never been disclosed.

[ REFERENCE ] to

[ patent document 1 ]: CN 108008132A;

[ patent document 2 ]: CN 103954761A.

Disclosure of Invention

The present invention relates generally to early ovarian cancer biomarkers, and in particular to methods and uses for predicting, assessing and diagnosing early ovarian cancer by measuring the levels of the biomarker combination Spondin1 and CA125, and also provides kits for predicting, assessing and diagnosing early ovarian cancer. The methods and kits provided by the present invention are particularly useful for epithelial ovarian cancer, and more particularly for early stage ovarian cancer (i.e., stage I or II ovarian cancer), thereby providing a method and kit that is easy to handle, highly sensitive, highly specific, and effective for early screening of ovarian cancer. In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:

in one aspect, the invention provides the use of an agent that detects levels of Spondin1 and CA125 in a sample from a subject in the manufacture of a kit for predicting, assessing or diagnosing early stage ovarian cancer in said subject. In some embodiments, the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer, or both stage I and stage II ovarian cancer.

In some embodiments, the reagents that detect levels of Spondin1 and CA125 are an antibody to Spondin1 and an antibody to CA125, respectively. Preferably, the antibody against Spondin1 and the antibody against CA125 are a monoclonal antibody against Spondin1 and a monoclonal antibody against CA125, respectively.

In some embodiments, the sample is a biological fluid sample from the subject. Preferably, the sample is blood, serum, plasma, lymph, cerebrospinal fluid, ascites, urine and tissue biopsy from the subject. More preferably, the sample is blood, serum and plasma from the subject.

In some embodiments, the kit is a chemiluminescent kit. Preferably, the chemiluminescent kit comprises a capture antibody, a detection antibody and a chemiluminescent substrate.

In some embodiments, the capture antibody is selected from the group consisting of a Spondin1 monoclonal antibody attached to a magnetic particle, a CA125 monoclonal antibody attached to a magnetic particle, and a combination thereof. Alternatively, the detection antibody is selected from the group consisting of a Spondin1 monoclonal antibody with an alkaline phosphatase label, a CA125 monoclonal antibody with an alkaline phosphatase label, and combinations thereof. Alternatively, the chemiluminescent substrate is AMPPD (CAS No: 122341-56-4).

In another aspect, the invention provides a kit for predicting, assessing or diagnosing ovarian cancer in a subject comprising reagents for detecting levels of Spondin1 and CA125 in a sample.

Preferably, the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer, or both stage I and stage II ovarian cancer.

In some embodiments, the reagents that detect levels of Spondin1 and CA125 are an antibody to Spondin1 and an antibody to CA125, respectively. Preferably, the antibody against Spondin1 and the antibody against CA125 are a monoclonal antibody against Spondin1 and a monoclonal antibody against CA125, respectively.

In some embodiments, the sample is a biological fluid sample from the subject. Preferably, the sample is blood, serum, plasma, lymph, cerebrospinal fluid, ascites, urine and tissue biopsy from the subject. More preferably, the sample is blood, serum and plasma from the subject.

In some embodiments, the kit is a chemiluminescent kit. In some embodiments, the kit is a diagnostic kit. Preferably, the chemiluminescent kit comprises a capture antibody, a detection antibody and a chemiluminescent substrate.

In some embodiments, the capture antibody is selected from the group consisting of a Spondin1 monoclonal antibody attached to a magnetic particle, a CA125 monoclonal antibody attached to a magnetic particle, and a combination thereof. Alternatively, the detection antibody is selected from the group consisting of a Spondin1 monoclonal antibody with an alkaline phosphatase label, a CA125 monoclonal antibody with an alkaline phosphatase label, and combinations thereof. Optionally, the chemiluminescent substrate is AMPPD.

In some embodiments, the kit comprises 2 reagent subgroups, wherein subgroup 1 comprises: magnetic particles coated with a monoclonal antibody against Spondin1, a Spondin1 monoclonal antibody with an alkaline phosphatase label, and a chemiluminescent substrate AMPPD; and is

Wherein subgroup 2 comprises: magnetic microparticles coated with a monoclonal antibody against CA125, a CA125 monoclonal antibody with an alkaline phosphatase label, and the chemiluminescent substrate AMPPD.

In another aspect of the present application, there is also provided a method of predicting, assessing or diagnosing the presence and stage of progression of ovarian cancer in a subject comprising detecting levels of Spondin1 and CA125 in a sample from the subject. In some embodiments, the levels of Spondin1 and CA125 in a sample of a subject can be detected by a kit as provided in the above aspects. Alternatively, the detected levels of Spondin1 and CA125 in a sample from a subject can be compared to values for Spondin1 and CA125 levels obtained from a non-diseased population (i.e., standard values) to determine the presence and stage of ovarian cancer in the subject.

Advantageous effects

The present invention uses Spondin1 and CA125 in combination as a novel marker for ovarian cancer screening and diagnosis. The inventors have demonstrated that there is a significant difference in the mean levels of Spondin1 and CA125 in the serum between the ovarian cancer patient group and the healthy control group (P < 0.01), and a correlation between the levels of Spondin1 and CA125 in the serum in the ovarian cancer patient group (r = 0.880). By jointly using the Spondin1 and the CA125 detection reagent as the markers for diagnosing ovarian cancer, compared with the method for singly detecting one of the markers, the sensitivity and specificity of the ovarian cancer diagnosis method and the kit provided by the invention are obviously improved, the false positive rate is greatly reduced, and the kit can be effectively used for early screening of ovarian cancer.

Drawings

In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:

FIG. 1 shows a standard curve for detecting Spondin1 protein by chemiluminescence.

FIG. 2 shows a standard curve for detecting CA125 protein by chemiluminescence method.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Some terms to which the present invention relates are explained as follows.

The biomarker "CA 125", carbohydrate antigen 125, also known as mucin-16, isMUC16A protein encoded by the gene. CA125 is one of the markers of ovarian epithelial cancer, and is widely used for diagnosis and follow-up of ovarian epithelial cancer at present. Herein, "CA 125" means a polypeptide biomarker or fragment thereof having at least 85% sequence identity to NCBI accession No. NP _ 078966.2.

The biomarker "Spondin 1", also known as F-Spondin, with accession number UniProtKB-Q9HCB6 in uniprot. Spondin1 is produced bySPON1The gene code, 807 amino acids in length, is a cell adhesion protein that promotes the attachment of spinal cord and sensory neuron cells and the growth of neurites in vitro. By similarity analysis, it is thought that it may contribute to the growth and guidance of axons in the spinal cord and peripheral nervous system. Herein, "Spondin 1" means a polypeptide biomarker or fragment thereof having at least 85% sequence identity to NCBI accession No. NP _ 006099. Specifically, the amino acid sequence of Spondin1 is shown as follows:

MRLSPAPLKLSRTPALLALALPLAAALAFSDETLDKVPKSEGYCSRILRAQGTRREGYTEFSLRVEGDPDFYKPGTSYRVTLSAAPPSYFRGFTLIALRENREGDKEEDHAGTFQIIDEEETQFMSNCPVAVTESTPRRRTRIQVFWIAPPAGTGCVILKASIVQKRIIYFQDEGSLTKKLCEQDSTFDGVTDKPILDCCACGTAKYRLTFYGNWSEKTHPKDYPRRANHWSAIIGGSHSKNYVLWEYGGYASEGVKQVAELGSPVKMEEEIRQQSDEVLTVIKAKAQWPAWQPLNVRAAPSAEFSVDRTRHLMSFLTMMGPSPDWNVGLSAEDLCTKECGWVQKVVQDLIPWDAGTDSGVTYESPNKPTIPQEKIRPLTSLDHPQSPFYDPEGGSITQVARVVIERIARKGEQCNIVPDNVDDIVADLAPEEKDEDDTPETCIYSNWSPWSACSSSTCDKGKRMRQRMLKAQLDLSVPCPDTQDFQPCMGPGCSDEDGSTCTMSEWITWSPCSISCGMGMRSRERYVKQFPEDGSVCTLPTEETEKCTVNEECSPSSCLMTEWGEWDECSATCGMGMKKRHRMIKMNPADGSMCKAETSQAEKCMMPECHTIPCLLSPWSEWSDCSVTCGKGMRTRQRMLKSLAELGDCNEDLEQVEKCMLPECPIDCELTEWSQWSECNKSCGKGHVIRTRMIQMEPQFGGAPCPETVQRKKCRIRKCLRNPSIQKLRWREARESRRSEQLKEESEGEQFPGCRMRPWTAWSECTKLCGGGIQERYMTVKKRFKSSQFTSCKDKKEIRACNVHPC

herein, the types of ovarian cancer predicted, evaluated or diagnosed are serous tumors, endometrioid tumors, mucinous tumors, and clear cell tumors. Also, predicting, assessing or diagnosing ovarian cancer includes predicting specific stages of the disease, such as stage I (IA, IB or IC), stage II, stage III and stage IV tumors. Additionally, "early stage of ovarian cancer" or "early stage ovarian cancer" means ovarian cancer that is in stage I or II. By "diagnosing" is meant identifying the presence or nature of a disorder (i.e., ovarian cancer). Diagnostic methods vary in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals in the population that test positive to total diseased individuals. The "specificity" of the diagnostic assay is 1 minus the false positive rate, where the "false positive" rate refers to the proportion of individuals who test positive but are not actually diseased.

By "chemiluminescent kit" is meant a kit for measuring the level of a biomarker using a chemiluminescent enzyme immunoassay (chlea). Specifically, in the chemiluminescent enzyme immunoassay, an immunoreaction is carried out with an enzyme-labeled bioactive substance, the enzyme on the immunoreactive complex acts on a luminescent substrate, emits light under the action of a signal reagent, and then is subjected to luminescence measurement with a luminescence signal measuring instrument, thereby quantitatively analyzing the level of a biomarker in a sample from a subject.

In some embodiments of the present application, the kits of the present application comprise a capture antibody, a detection antibody, and a chemiluminescent substrate. Specifically, the capture antibody can be prepared by linking a monoclonal antibody against the target biomarker to magnetic microparticles in the presence of a coupling solution under appropriate reaction conditions. Advantageously, the capture antibody can be obtained by preparing magnetic particles coated with murine anti-human Spondin1 monoclonal antibody by coating the magnetic particles with an appropriate concentration of murine anti-human Spondin1 monoclonal antibody at 37 ℃ for 2 hours or at 4 ℃ overnight in the presence of a coupling solution.

Further, the detection antibody may be a Spondin1 monoclonal antibody with an alkaline phosphatase label, a CA125 monoclonal antibody with an alkaline phosphatase label, and combinations thereof. Optionally, the chemiluminescent substrate is AMPPD.

It should be noted that the level of the biomarker in the sample can also be detected by other methods known in the art, such as but not limited to enzyme-linked immunosorbent assay (ELISA), immunofluorescence chromatography, electrochemiluminescence, etc., and the invention is not limited thereto.

Examples

The purchase information of the products used in the examples is as follows, but is not limited to the same manufacturer.

Murine anti-human Spondin1 monoclonal antibody (coating antibody): the goods number is: AF3135, available from R & D under the trade name SPON1 Antibody.

Mouse anti-human Spondin1 monoclonal antibody (detection antibody): the goods number is: sc-390182, available from SANTA CRUZ under the trade name F-spondin antibody.

Magnetic particles: the goods number is: MagCOOH, under the trade name carboxyl magnetic beads, was purchased from knoyi microsphere technologies, inc.

Spondin1 protein standard: the goods number is: 3135-SP-025/CF, available from R & D under the trade name SPON1 protein.

Mouse anti-human CA125 monoclonal antibody (coating antibody), cat #: CA125-McAb2, purchased from Fipeng organisms under the trade name carbohydrate antigen 125 antibody.

Mouse anti-human CA125 monoclonal antibody (detection antibody), cat #: CA125-McAb1, purchased from Fipeng organisms under the trade name carbohydrate antigen 125 antibody.

Standard CA125 protein: the goods number is: CA125-Ag, available from Fipeng organisms under the trade name carbohydrate antigen 125.

Alkaline phosphatase (alkaline phosphatase): the goods number is: SP011401, sold under the trade name alkaline phosphatase, was purchased from national reagent.

AMPPD: CAS No. 122341-56-4, 4-methoxy-4- (3-phosphorylphenyl) spiro [1, 2-dioxetane-3, 2' -adamantane ], is an alkaline phosphatase substrate, commercially available from kyoto jenko jiekang under the trade name chemiluminescence substrate.

Tween-20: commercially available from biologies under the trade name Tween-20.

Example 1 establishment of Spondin1 detection System

Constructing a detection system: coating the magnetic microparticles with the mouse anti-human Spondin1 monoclonal antibody at a concentration of 2.5. mu.g/mL for 2 hours at 37 ℃ or overnight at 4 ℃ to prepare magnetic microparticles coated with the mouse anti-human Spondin1 monoclonal antibody, i.e., capture antibodies; adding Spondin1 protein standard substances with the concentrations of 8000, 4000, 2000, 1000, 500 and 0 pg/ml into different reaction cups respectively, and reacting for 30 minutes at 37 ℃; then adding 0.25 mug/mL alkaline phosphatase labeled mouse anti-human Spondin1 monoclonal antibody (namely, detection antibody), reacting for 30 minutes at 37 ℃, washing magnetic beads, and removing supernatant; finally, a luminescent substrate, AMPPD, was added and the luminescence value was measured. According to the luminescence values of the reaction system obtained from the standard substance values with different concentrations, a Spondin1 protein standard curve is constructed, and the result is shown in fig. 1. As can be seen from FIG. 1, the linear range of the Spondin1 detection system is 500 pg/mL-8000 pg/mL, the linear correlation coefficient r of the standard substance in the linear range is more than or equal to 0.990, and the recovery rate is in the range of 90% -110%.

Determining a detection system: the antibody with different concentrations is detected by a checkerboard matrix method, wherein the capture antibody is selected from 0.5, 1.5, 2.5, 3.5, 4.5 and 5.5 mu g/mL, and the detection antibody is selected from 0.05, 0.15, 0.2, 0.25, 0.35, 0.45 and 0.55 mu g/mL. When the antibodies with different concentrations are detected, the optimal working concentration of the Spondin1 capture antibody is 2.5 mu g/mL, and the optimal working concentration of the Spondin1 detection antibody is 0.25 mu g/mL.

Example 2 Spondin1 chemiluminescence detection kit

A Spondin1 chemiluminescence assay kit was constructed according to the Spondin1 serum assay system in example 1, and the specific components are shown in Table 1:

TABLE 1 Spondin1 chemiluminescence assay kit Components

Evaluation of Spondin1 chemiluminescence detection kit: detecting the Spondin1 repetitive reference substance by using a Spondin1 chemiluminescence detection kit, wherein the Spondin1 repetitive reference substance is repeatedly detected for 10 times at two levels of the Spondin1 protein concentration of 2000 pg/mL and 1000 pg/mL respectively, and the result shows that the coefficient of variation CV is less than or equal to 10%; when 3 batch number kits are used for detecting the same sample, the inter-batch variation coefficient CV of the 3 batch number kits is less than or equal to 15 percent.

Example 3 establishment of CA125 serum detection reaction System and optimization thereof

Coating the magnetic beads with a mouse anti-human CA125 monoclonal antibody with the concentration of 5 mu g/mL, and coating overnight at 37 ℃ for 2 hours or 4 ℃; respectively adding the CA125 protein standard substance and the serum sample with the concentrations of 0U/mL, 10U/mL, 20U/mL, 40U/mL, 80U/mL, 160U/mL and 320U/mL into the reaction holes, and reacting for 30 minutes at 37 ℃; then, a mouse anti-human CA125 monoclonal antibody marked by alkaline phosphatase with the concentration of 0.5 mu g/mL is used for reacting for 30 minutes at the temperature of 37 ℃, and the magnetic beads are washed to remove the supernatant; finally, a luminescent substrate, AMPPD, was added and the luminescence value was measured. A calibration curve of CA125 was constructed based on the luminescence values of the reaction system obtained from the standard values of different concentrations, and the results are shown in FIG. 2. As can be seen from FIG. 2, the linear range of the CA125 chemiluminescence detection kit is 10U/mL-320U/mL, the linear correlation coefficient r of the standard substance in the linear range is more than or equal to 0.990, and the recovery rate is in the range of 90% -110%.

Determining a detection system: similar to the checkerboard method in example 2, the best working concentration of the home-made CA125 capture antibody was found to be 5. mu.g/mL and the best working concentration of the CA125 detection antibody was found to be 0.5. mu.g/mL by detecting different concentrations of the antibody.

The main components of the detection system are determined through the research, and then a CA125 serum detection system is established.

Example 4 CA125 chemiluminescence detection kit

The CA125 chemiluminescence assay kit is constructed according to the CA125 serum assay system established in the example 3, and the specific components are shown in the following table 2:

TABLE 2 CA125 chemiluminescence detection kit components

Evaluation of CA125 chemiluminescent assay kit: detecting a CA125 repetitive reference substance by using a CA125 chemiluminescence detection kit, and repeatedly detecting for 10 times at two levels of 20U/mL and 80 pg/mL of CA125 protein concentration respectively, wherein the result shows that the coefficient of variation CV is less than or equal to 10%; when 3 batch number kits are used for detecting the same sample, the inter-batch variation coefficient CV of the 3 batch number kits is less than or equal to 15 percent.

Example 5 Spondin 1-CA125 Combined detection kit diagnoses and predicts early ovarian cancer

100 clinical samples were collected, 22 samples from the normal population and 78 samples from early stage ovarian cancer patients, each with 1 mL serum. Of the 78 ovarian cancer patients, 24 were stage I ovarian cancer patients and 54 were stage II ovarian cancer patients. Respectively detecting the concentrations of Spondin1 and CA125 markers in blood serum of ovarian cancer patients and healthy normal people, analyzing the obtained data by using SPASS statistical software according to the detection result, and if the difference between different groups has statistical significance, performing independent sample t test, wherein the result is expressed by x +/-s; the association analysis of Spondin1 and CA125 in each group is statistically significant with P <0.05 as the difference. Finally, the specificity and sensitivity of the Spondin1 and CA125 markers were measured individually or in combination according to data statistics, and the results are shown in table 3.

The results show that the serum CA125 (591.96 + -384.5) U/mL and Spondin1 (3114.51 + -105.49) pg/mL in the ovarian cancer group are both higher than those in the healthy control group CA125 (30.45 + -25.87) U/mL and Spondin1 (2277.04 + -95.35) pg/mL. Statistical treatment was performed and the differences were statistically significant (p < 0.01) and the results are shown in table 3 below.

TABLE 3 serum CA125 and Spondin1 assay results (X + -S) for ovarian cancer group and healthy control group

Note: compared with the healthy control group, the ovarian cancer group has P less than 0.05; comparison of serum CA125 between ovarian cancer and healthy controls, P <0.05, and comparison of serum Spondin1 levels between ovarian cancer and healthy controls, P < 0.05.

Meanwhile, there was a correlation between the ovarian cancer group CA125 and Spondin1 levels (r = 0.880), while there was no significant correlation between the healthy control group CA125 and Spondin1 levels (r = 0.240). The results are shown in Table 4 below.

TABLE 4 correlation between CA125 and Spondin1 in ovarian cancer and healthy control groups

Note: there was a significant correlation between the ovarian cancer group CA125 and Spondin1 levels (r = 0.880), while there was no significant correlation between the healthy control group CA125 and Spondin1 levels (r = 0.240).

The sensitivity and specificity analysis of the ovarian cancer diagnosis case can be known as follows: the sensitivity of detecting the Spondin1 marker alone is 74.3%, and the specificity is 87%; the sensitivity of the CA125 marker alone was 82.5% and the specificity was 70.1%. The sensitivity was 84.7% and the specificity was 91.4% when both Spondin1 and CA125 markers were tested in combination, the results are shown in Table 5 below.

TABLE 5 ovarian cancer diagnosis results of Spondin 1-CA125 combined detection kit

Note: the detection of the combination of the Spondin1 marker and the CA125 marker is obviously superior to the detection of a single ovarian cancer marker in terms of sensitivity and specificity.

Therefore, the combined detection result of the Spondin1 marker and the CA125 marker is obviously superior to that of a single ovarian cancer marker. In conclusion, Spondin1 and CA125 can be used as markers for diagnosing and predicting early ovarian cancer, can be used for early diagnosis of ovarian cancer, and improves accuracy of early prediction.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims (8)

1. Use of an agent that detects levels of Spondin1 and CA125 in a sample from a subject in the manufacture of a kit for predicting, assessing or diagnosing ovarian cancer in said subject, wherein said ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer or both stage I and stage II ovarian cancer, and said sample is blood, serum, plasma from said subject,

wherein, the reagent for detecting the levels of the Spondin1 and the CA125 is an antibody aiming at the Spondin1 and an antibody aiming at the CA125 respectively.

2. The use according to claim 1, wherein said antibody directed against Spondin1 and antibody directed against CA125 are monoclonal antibody directed against Spondin1 and monoclonal antibody directed against CA125, respectively.

3. Use according to claim 1, wherein the kit is a chemiluminescent kit comprising a capture antibody, a detection antibody and a chemiluminescent substrate.

4. The use according to claim 3, wherein the capture antibody is selected from the group consisting of a Spondin1 monoclonal antibody attached to a magnetic particle, a CA125 monoclonal antibody attached to a magnetic particle, and combinations thereof;

the detection antibody is selected from a Spondin1 monoclonal antibody with an alkaline phosphatase label, a CA125 monoclonal antibody with an alkaline phosphatase label, and a combination thereof; and/or the presence of a gas in the gas,

the chemiluminescent substrate is AMPPD.

5. A kit for predicting, assessing or diagnosing ovarian cancer in a subject comprising reagents for detecting the levels of Spondin1 and CA125 in a sample, wherein the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer or both stage I and stage II ovarian cancer, and the sample is blood, serum, plasma from the subject,

wherein, the reagent for detecting the levels of the Spondin1 and the CA125 is an antibody aiming at the Spondin1 and an antibody aiming at the CA125 respectively.

6. The kit according to claim 5, wherein the antibody against Spondin1 and the antibody against CA125 are a monoclonal antibody against Spondin1 and a monoclonal antibody against CA125, respectively.

7. The kit of claim 5, wherein the kit is a chemiluminescent kit comprising a capture antibody, a detection antibody, and a chemiluminescent substrate, wherein:

the capture antibody is selected from the group consisting of a Spondin1 monoclonal antibody linked to the magnetic particle, a CA125 monoclonal antibody linked to the magnetic particle, and combinations thereof;

the detection antibody is selected from a Spondin1 monoclonal antibody with an alkaline phosphatase label, a CA125 monoclonal antibody with an alkaline phosphatase label, and a combination thereof; and/or the presence of a gas in the gas,

the chemiluminescent substrate is AMPPD.

8. The kit according to claim 5, characterized in that it comprises 2 subgroups of reagents,

wherein subgroup 1 comprises: magnetic particles coated with a monoclonal antibody against Spondin1, a Spondin1 monoclonal antibody with an alkaline phosphatase label, and a chemiluminescent substrate AMPPD; and is

Wherein subgroup 2 comprises: magnetic microparticles coated with a monoclonal antibody against CA125, a CA125 monoclonal antibody with an alkaline phosphatase label, and the chemiluminescent substrate AMPPD.

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