CN111693703A - Spondin1 and HE4 combined 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 HE4, belonging to the technical field of immunodetection. The invention discloses the use of reagents for detecting the levels of Spondin1 and HE4 in a sample from a subject in the manufacture of a kit for predicting, assessing or diagnosing early stage ovarian cancer in said 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 HE4 combined 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 HE4 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 attack, the early diagnosis of the ovarian cancer is difficult, and the technical problem is solved. In clinical findings, it has often progressed to the middle and advanced stages. 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 ovarian cancer markers in blood. For ovarian cancer markers, although the gynecological oncology division of the Chinese medical society recommends the combined application of HE4 and CA125, 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 HE4 and CA125 detection was not improved in stage I ovarian cancer (see Moore RG, Brown AK, Miller MC et al, The use of multiple novel biological markers for The detection of ovarian cancer in patients with a viral mass. Gynecoloncol,2008,108: 402-408).

Therefore, there is still a lack of accurate and reliable tumor markers for early diagnosis of ovarian cancer, and there is an urgent need to develop some new serum markers for ovarian cancer with diagnostic value. The ideal marker needs to be able to be sensitively and specifically detected from the peripheral blood in the early stages of ovarian cancer cachexia.

Most of the commercially available kits use ELISA method, and the principle is as follows: coating the CA125 antibody in a 96-hole microporous plate to prepare a solid phase carrier, respectively adding a standard substance or a specimen into the micropores, wherein the CA125 is combined with the antibody connected to the solid phase carrier, then adding biotinylated CA125 antibody, washing the unbound biotinylated antibody, adding HRP-labeled avidin, thoroughly washing again, and adding TMB substrate for color development. TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid. The shade of the color was positively correlated with the CA125 in the sample. The absorbance (o.d. value) was measured at a wavelength of 450nm with a microplate reader, and the sample concentration was calculated. However, this method is subjectively influenced by a large operator. It is desirable to provide a test kit that reduces human error.

The Spondin1 protein (accession number UniProtKB-Q9HCB6 in uniprot. org), also known as F-Spondin, encoded by the SPON1 gene in humans, is 807 amino acids in length, is a cell adhesion protein that promotes spinal cord and sensory neuron cell attachment and neurite growth 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 HE4 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 HE4, 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 HE4 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 HE4 are an antibody to Spondin1 and an antibody to HE4, respectively. Preferably, the antibody directed to Spondin1 and the antibody directed to HE4 are a monoclonal antibody directed to Spondin1 and a monoclonal antibody directed to HE4, 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 the magnetic particle, a HE4 monoclonal antibody attached to the 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 HE4 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 HE4 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 HE4 are an antibody to Spondin1 and an antibody to HE4, respectively. Preferably, the antibody directed to Spondin1 and the antibody directed to HE4 are a monoclonal antibody directed to Spondin1 and a monoclonal antibody directed to HE4, 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 the magnetic particle, a HE4 monoclonal antibody attached to the 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 HE4 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, Spondin1 monoclonal antibody with alkaline phosphatase label, and chemiluminescent substrate AMPPD; and is

Wherein subgroup 2 comprises: magnetic microparticles coated with a monoclonal antibody against HE4, HE4 monoclonal antibody with 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 HE4 in a sample from the subject. In some embodiments, the levels of Spondin1 and HE4 in a sample from a subject can be detected by a kit as provided in the above aspects. Alternatively, the detected levels of Spondin1 and HE4 in a sample from the subject can be compared to values for Spondin1 and HE4 levels (i.e., standard values) obtained from a non-diseased population to determine the presence and stage of ovarian cancer in the subject.

Advantageous effects

The invention uses Spondin1 and HE4 in combination as a novel marker for ovarian cancer screening and diagnosis. The inventor has proved that by using Spondin1 and HE4 detection reagents in combination as markers for diagnosing ovarian cancer, compared with the method for detecting one of the markers alone, the sensitivity and specificity of the ovarian cancer diagnosis method and the kit provided by the invention are both obviously improved (the sensitivity is 87.1% and the specificity is 91.2%), the false positive rate is greatly reduced, and a method for screening early ovarian cancer with low misdiagnosis rate, reduced human error and improved accuracy and a corresponding chemiluminescence detection kit are provided.

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 detection of HE4 protein by chemiluminescence.

FIG. 3 shows the ROC curve of the Spondin 1-HE4 combined test kit for ovarian cancer diagnosis.

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 "HE 4", Human epididymis secreted protein (Human epididymis secreted protein e4, HE4) belongs to the whey acidic 4-disulfide center (WFDC) protein family, and has the characteristics of a suspected trypsin inhibitor. HE4 is a secreted glycoprotein discovered by Kirchhoff et al in epididymis epithelial tissue in 1991, is a protease inhibitor in sperm maturation, and is also expressed in normal women. Early diagnosis of ovarian cancer is currently performed using HE4 or its combination with CA 125. Herein, biomarker "HE 4" means a polypeptide biomarker or fragment thereof having at least 85% sequence identity to NCBI accession number CAA 44869.

The biomarker "Spondin 1", also known as F-Spondin, with accession number UniProtKB-Q9HCB6 in uniprot. Spondin1 is encoded by the SPON1 gene in humans, is 807 amino acids in length, is a cell adhesion protein that promotes spinal cord and sensory neuron cell attachment and neurite outgrowth 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.

"Chemiluminescent kit" means a kit for measuring the level of a biomarker using a Chemiluminescent enzyme immunoassay (cLEIA). 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 HE4 monoclonal antibody with an alkaline phosphatase label, and combinations thereof. Optionally, the chemiluminescent substrate is AMPPD.

It is noted that although the level of the biomarker in the sample may be detected by other methods known in the art, such as, but not limited to, enzyme linked immunosorbent assay (ELISA), immunofluorescence chromatography, electrochemiluminescence, etc., the present inventors have unexpectedly discovered that when combining chemiluminescence with the biomarkers Spondin1 and HE4 of the present application, at least the following advantages are achieved:

1. early stage ovarian cancer patients are detected with high sensitivity (e.g., 10-22 mol/L). This is far above the detection limit of other immunoassay methods, such as radioimmunoassay and enzyme-linked immunoassay.

2. Has wide linear dynamic range. In the methods and kits of the present application, the luminescence intensity is in a linear relationship between 4 and 6 orders of magnitude with the concentration of the substance being measured. This range is much greater than the linear range of absorbance (OD values) in the enzyme immunoassay (2.0).

3. The result is stable, the error is small, the sample directly emits light without any light source irradiation, the influence of a plurality of possible factors (light source stability, light scattering, optical wave selector and the like) on the analysis is eliminated, and the analysis is sensitive, stable and reliable.

Thus, the combination of the chemiluminescent method with the biomarkers Spondin1 and HE4 of the present application has the advantages described above.

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, purchased 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, commercially available 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.

A murine anti-human HE4 monoclonal antibody (coating antibody), cat # HE4-McAb1, purchased from a ficoll organism under the trade name HE 4.

A murine anti-human HE4 monoclonal antibody (detection antibody), cat # HE4-McAb2, purchased from a ficoll organism under the trade name HE 4.

HE4 protein standard: cat No. HE4-Ag, available from Fipeng organisms under the trade name HE 4.

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 and optimization thereof

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 0pg/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 500pg/mL-8000pg/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 chemiluminescent 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 2000pg/mL and 1000pg/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 HE4 serum detection reaction System and optimization thereof

Coating the magnetic beads with a mouse anti-human HE4 monoclonal antibody with the concentration of 5 mu g/mL, and coating overnight at 37 ℃ for 2 hours or 4 ℃; then adding the HE4 protein standard and the serum sample with the concentration of 0pmol/L, 20pmol/L, 40pmol/L, 80pmol/L, 160pmol/L, 320pmol/L and 640pmol/L into the closed plate respectively, and reacting for 30 minutes at 37 ℃; then using a mouse anti-human HE4 monoclonal antibody with the concentration of 0.5 mu g/mL alkaline phosphatase for reaction for 30 minutes at the temperature of 37 ℃, and cleaning magnetic beads to remove supernatant; finally, a luminescent substrate, AMPPD, was added and the luminescence value was measured. A calibration curve of HE4 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 HE4 chemiluminescence detection kit is 20pmol/L-640pmol/L, the linear correlation coefficient r of the standard substance is more than or equal to 0.990 in the linear range, and the recovery rate is in the range of 90% -110%.

Determining a detection system: similar to the checkerboard method in example 2, the optimal working concentration of the HE4 capture antibody is 5 μ g/mL and the optimal working concentration of the HE4 detection antibody is 0.5 μ g/mL by detecting different concentrations of the antibody.

The main components of the detection system are determined through the research, and then an HE4 serum detection system is established.

Example 4 HE4 chemiluminescence detection kit

An HE4 chemiluminescence assay kit is constructed according to the HE4 serum assay system established in example 3, and the specific components are shown in the following table 2:

TABLE 2 HE4 chemiluminescence assay kit Components

Evaluation of HE4 chemiluminescent assay kit: detecting HE4 repetitive reference substance by using an HE4 chemiluminescence detection kit, and repeatedly detecting for 10 times at two levels of HE4 protein concentration of 40pmol/L and 80pmol/L 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 human ovarian cancer marker Spondin 1-HE4 Combined detection kit

The Spondin1 chemiluminescence detection kit constructed in example 2 and the HE4 chemiluminescence detection kit constructed in example 4 were combined to form a Spondin 1-HE4 combined detection kit.

Example 6 Spondin 1-HE4 Combined test kit diagnosis and prognosis of ovarian cancer

100 clinical samples were collected, 22 samples from the normal population and 78 samples from early stage ovarian cancer patients, each with 1mL serum. Of the 78 ovarian cancer patients, 24 were stage I ovarian cancer patients and 54 were stage II ovarian cancer patients. The concentrations of Spondin1 and HE4 markers in the serum of ovarian cancer patients and healthy normal persons were respectively detected, and ROC curves were drawn according to the detection results (FIG. 3). The results show that the cutoff (cut off) value for Spondin1 to differentiate healthy humans from ovarian cancer patients is 2670pg/mL, and the cutoff value for HE4 to differentiate healthy humans from ovarian cancer patients is 67.25 pmol/L. The area under the curve, specificity and sensitivity of the individual or combined detection of Spondin1 and HE4 markers were then determined according to ROC curve statistics and the results are shown in table 3.

The results show that the serum HE4(591.96 +/-384.5) U/mL and Spondin 1(3114.51 +/-105.49) pg/mL of the ovarian cancer group are both higher than that of HE4(30.45 +/-25.87) U/mL and Spondin 1(2277.04 +/-95.35) pg/mL of the healthy control group. Statistical treatment was performed and the differences were statistically significant (p <0.01) and the results are shown in table 3 below.

TABLE 3 ovarian cancer diagnosis results of Spondin 1+ HE4 combined detection kit

Marker substance	Area under curve	Sensitivity of the probe	Specificity of
				Spondin 1	0.782	74.3％	87％
HE4	0.854	77.1％	80％
				Spondin 1+HE4	0.921	87.1％	91.2％

Note: the detection by combining the Spondin1 marker and the HE4 marker is obviously superior to the detection of a single ovarian cancer marker in terms of sensitivity and specificity.

As can be seen from Table 3, the area under the curve for detecting the Spondin1 marker alone is 0.782, the sensitivity is 74.3%, and the specificity is 87%; the area under the curve for the HE4 marker detected alone was 0.854, the sensitivity was 77.1%, and the specificity was 80%. When the two markers Spondin1 and HE4 are detected in a combined manner, the area under the curve is 0.921, the sensitivity is 87.1 percent, and the specificity is 91.2 percent. Therefore, the combined detection of the two markers Spondin1 and HE4 is obviously superior to the detection of a single ovarian cancer marker.

In conclusion, Spondin1 and HE4 can be used as markers for diagnosing and predicting early ovarian cancer, can be used for early diagnosis of ovarian cancer, and can improve the 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 (10)

1. Use of an agent that detects levels of Spondin1 and HE4 in a sample from a subject, wherein the ovarian cancer is selected from stage I ovarian cancer, stage II ovarian cancer or both stage I and stage II ovarian cancer, in the manufacture of a kit for predicting, assessing or diagnosing ovarian cancer in the subject, the sample being blood, serum, plasma from the subject.

2. The use of claim 1, wherein the reagents for detecting levels of Spondin1 and HE4 are an antibody against Spondin1 and an antibody against HE4, respectively.

3. The use according to claim 2, wherein said antibody directed against Spondin1 and antibody directed against HE4 are monoclonal antibody directed against Spondin1 and monoclonal antibody directed against HE4, respectively.

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

5. The use according to claim 4, wherein the capture antibody is selected from the group consisting of a Spondin1 monoclonal antibody linked to a magnetic particle, a HE4 monoclonal antibody linked to a magnetic particle, and combinations thereof;

the detection antibody is selected from a Spondin1 monoclonal antibody with an alkaline phosphatase label, a HE4 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.

6. A kit for predicting, assessing or diagnosing ovarian cancer in a subject comprising reagents for detecting the levels of Spondin1 and HE4 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.

7. The kit of claim 6, wherein the reagents for detecting levels of Spondin1 and HE4 are an antibody against Spondin1 and an antibody against HE4, respectively.

8. The kit of claim 7, wherein said antibody directed to Spondin1 and said antibody directed to HE4 are a monoclonal antibody directed to Spondin1 and a monoclonal antibody directed to HE4, respectively.

9. The kit of claim 6, 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 attached to the magnetic particle, a HE4 monoclonal antibody attached to the magnetic particle, and combinations thereof;

the detection antibody is selected from a Spondin1 monoclonal antibody with an alkaline phosphatase label, a HE4 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.

10. The kit of claim 6, wherein 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 HE4, HE4 monoclonal antibody with alkaline phosphatase label, and the chemiluminescent substrate AMPPD.

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