CN111693703B - Spondin1 and HE4 combined as early ovarian cancer biomarker and kit - Google Patents

Abstract

The invention relates to a combination of Spondin1 and HE4 as an early ovarian cancer biomarker and a kit, belonging to the technical field of immunodetection. The present invention discloses the use of reagents for detecting levels of Spondin1 and HE4 in a sample from a subject in the preparation of a kit for predicting, evaluating or diagnosing early stage ovarian cancer in said subject. The invention also provides a kit for predicting, evaluating or diagnosing the early ovarian cancer of a subject, which can be effectively used for early screening of the ovarian cancer, and the sensitivity and specificity of the prediction, evaluation and diagnosis of the ovarian cancer in the subject are obviously improved and the false positive rate in early ovarian cancer detection is greatly reduced by the kit provided by the invention.

Description

Spondin1 and HE4 combined as early ovarian cancer biomarker and kit

Technical Field

The present invention relates generally to the field of immunodetection technology, and in particular to the use of a combination of Spondin1 and HE4 as biomarkers for early ovarian cancer, and kits made therefrom.

Background

Ovarian cancer is one of the common malignant tumors of female reproductive organs, and causes serious threat to female life. The ovarian is deep in the pelvic cavity, small in size and lack of typical symptoms during onset, so that early diagnosis of ovarian cancer is difficult, and the early diagnosis becomes a technical problem. Clinical findings have often progressed to mid-to late-stages. And to middle and late stages, particularly after metastasis, even when treated by ovarian epithelial cancer surgery, the recurrence rate and 5-year survival rate are low. Thus, early diagnosis of ovarian cancer is important for reducing metastasis and improving 5-year survival rate. Currently, the diagnosis of ovarian cancer is based on vaginal ultrasound (TVU) and detection of ovarian cancer markers in the blood. For ovarian cancer markers, although the Chinese society of medical science, the department of gynecology, oncology recommended the combined use of HE4 and CA125, the sensitivity of single HE4 to the auxiliary diagnosis of stage I ovarian cancer was higher than that of CA125, but was 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 tumor biomarkers for the detection of ovarian carcinoma in patients with a pelvic mass. Gynecol Oncol,2008, 108:402-408).

Thus, there is still a lack of accurate and reliable tumor markers for early diagnosis of ovarian cancer, and development of new serum markers for ovarian cancer with diagnostic value is urgently needed. The ideal marker needs to be able to be detected sensitively and specifically from peripheral blood at the early stages of ovarian cancer malignancy.

Most of the commercial kits adopt ELISA method, the principle is as follows: coating CA125 antibody in 96-well micro-pore plate to prepare solid phase carrier, adding standard substance or specimen into micro-pore, respectively, wherein CA125 is combined with antibody connected to the solid phase carrier, then adding biotinylated CA125 antibody, washing unbound biotinylated antibody, adding HRP-labeled avidin, thoroughly washing again, and adding TMB substrate for color development. TMB is converted to blue under the catalysis of peroxidase and to a final yellow color under the action of acid. The shade of the color and CA125 in the sample were positively correlated. The absorbance (o.d. value) was measured at a wavelength of 450nm using an enzyme-labeled instrument, and the sample concentration was calculated. However, this method is subject to great subjective impact by the operator. It is desirable to provide a detection kit that reduces human error.

The Spondin1 protein (UniProtKB-Q9 HCB6 in uniprotrot. Org), also known as F-Spondin, is encoded by the SPON1 gene in humans and is 807 amino acids in length, a cell adhesion protein that promotes the attachment of spinal cord and sensory neuronal cells and neurite outgrowth in vitro. Through 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 use of HE4 and Spondin1 in combination as biomarkers for early ovarian cancer diagnosis has never been disclosed.

[ reference ] to

[ patent document 1]: CN108008132a;

[ patent document 2]: CN103954761a.

Disclosure of Invention

The present invention relates generally to early ovarian cancer biomarkers and in particular to methods and uses for predicting, evaluating and diagnosing early ovarian cancer by measuring the levels of the biomarker combinations Spondin1 and HE4, and also provides kits for predicting, evaluating and diagnosing early ovarian cancer. The methods and kits provided herein are particularly useful for epithelial ovarian cancer, and more particularly for early stage ovarian cancer (i.e., stage I or stage II ovarian cancer), thereby providing a method and kit that is easy to handle, highly sensitive, highly specific, and can be effectively used for early screening of ovarian cancer. In order to achieve the above object 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 preparation of a kit for predicting, evaluating or diagnosing early stage ovarian cancer in the 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 agent that detects the levels of Spondin1 and HE4 is an antibody directed against Spondin1 and an antibody directed against HE4, respectively. Preferably, the antibodies to Spondin1 and to HE4 are a monoclonal antibody to Spondin1 and a monoclonal antibody 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 biopsies 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 linked to magnetic particles, a HE4 monoclonal antibody linked to magnetic particles, and combinations 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, evaluating 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 agent that detects the levels of Spondin1 and HE4 is an antibody directed against Spondin1 and an antibody directed against HE4, respectively. Preferably, the antibodies to Spondin1 and to HE4 are a monoclonal antibody to Spondin1 and a monoclonal antibody 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 biopsies 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 linked to magnetic particles, a HE4 monoclonal antibody linked to magnetic particles, and combinations 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.

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

Wherein subgroup 2 comprises: magnetic particles coated with a monoclonal antibody directed against HE4, HE4 monoclonal antibody with alkaline phosphatase label, and chemiluminescent substrate AMPPD.

In another aspect of the present application, there is also provided a method of predicting, evaluating or diagnosing the presence of ovarian cancer and stage of its development 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 of 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 of the subject can be compared to the values (i.e., standard values) of levels of Spondin1 and HE4 obtained from the non-diseased population to determine the presence and stage of ovarian cancer in the subject.

Advantageous effects

The present invention uses Spondin1 and HE4 in combination as novel markers for ovarian cancer screening and diagnosis. The inventors have demonstrated that by combining the Spondin1 and HE4 detection reagents as markers for diagnosing ovarian cancer, the sensitivity and specificity of the ovarian cancer diagnosis method and kit provided by the invention are both significantly improved (the sensitivity is 87.1% & specificity is 91.2%) compared to the detection of one of the markers alone, greatly reducing the false positive rate, and providing a method for early ovarian cancer screening with low misdiagnosis rate, reduced human error and improved accuracy, and a corresponding chemiluminescent detection kit.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention clearer, the present invention provides the following drawings:

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

FIG. 2 shows a standard curve for chemiluminescent detection of HE4 protein.

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

Detailed Description

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

The biomarker "HE4", human epididymal secretion protein (Human epididymis secretory protein E, HE 4), belongs to the whey acidic 4-disulfide center (WFDC) protein family, with the property of being suspected of being a trypsin inhibitor. HE4 is a secreted glycoprotein found in epididymal epithelial tissue by Kirchhoff et al in 1991, is a protease inhibitor during sperm maturation, and is expressed in normal females. HE4 or its combination with CA125 is currently used for early diagnosis of ovarian cancer. In this context, biomarker "HE4" 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, is designated uniProtKB-Q9HCB6 in uniprotrot.org. Spondin1 is encoded by the SPON1 gene in humans and is 807 amino acids in length, a cell adhesion protein that promotes the attachment of spinal cord and sensory neuron cells and the growth of neurites in vitro. Through similarity analysis, it is thought that it may contribute to the growth and guidance of axons in the spinal cord and peripheral nervous system. In this context, "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 below:

MRLSPAPLKLSRTPALLALALPLAAALAFSDETLDKVPKSEGYCSRILRAQGTRREGYTEFSLRVEGDPDFYKPGTSYRVTLSAAPPSYFRGFTLIALRENREGDKEEDHAGTFQIIDEEETQFMSNCPVAVTESTPRRRTRIQVFWIAPPAGTGCVILKASIVQKRIIYFQDEGSLTKKLCEQDSTFDGVTDKPILDCCACGTAKYRLTFYGNWSEKTHPKDYPRRANHWSAIIGGSHSKNYVLWEYGGYASEGVKQVAELGSPVKMEEEIRQQSDEVLTVIKAKAQWPAWQPLNVRAAPSAEFSVDRTRHLMSFLTMMGPSPDWNVGLSAEDLCTKECGWVQKVVQDLIPWDAGTDSGVTYESPNKPTIPQEKIRPLTSLDHPQSPFYDPEGGSITQVARVVIERIARKGEQCNIVPDNVDDIVADLAPEEKDEDDTPETCIYSNWSPWSACSSSTCDKGKRMRQRMLKAQLDLSVPCPDTQDFQPCMGPGCSDEDGSTCTMSEWITWSPCSISCGMGMRSRERYVKQFPEDGSVCTLPTEETEKCTVNEECSPSSCLMTEWGEWDECSATCGMGMKKRHRMIKMNPADGSMCKAETSQAEKCMMPECHTIPCLLSPWSEWSDCSVTCGKGMRTRQRMLKSLAELGDCNEDLEQVEKCMLPECPIDCELTEWSQWSECNKSCGKGHVIRTRMIQMEPQFGGAPCPETVQRKKCRIRKCLRNPSIQKLRWREARESRRSEQLKEESEGEQFPGCRMRPWTAWSECTKLCGGGIQERYMTVKKRFKSSQFTSCKDKKEIRACNVHPC

in this context, the types of ovarian cancer predicted, assessed or diagnosed are serous, endometrioid, mucinous and clear cell tumors. Also, predicting, evaluating or diagnosing ovarian cancer includes predicting a particular stage of a 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 in stage I or stage II. By "diagnosis" 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 individuals with positive disease in a population that are tested to be total individuals with disease. The "specificity" of a diagnostic assay is 1 minus the false positive rate, where "false positive" rate refers to the proportion of individuals tested positive but not actually diseased.

By "chemiluminescent kit" is meant a kit for determining the level of a biomarker using a chemiluminescent enzyme immunoassay (Chemiluminescent enzyme immunoassay, cLEIA). Specifically, in chemiluminescent enzyme immunoassay, an enzyme is used for labeling a bioactive substance for immune reaction, enzyme on an immune reaction complex acts on a luminescent substrate again, luminescence is generated under the action of a signal reagent, and then luminescence measurement is performed by a luminescence signal measuring instrument, so that the biomarker level in a sample from a subject is quantitatively analyzed.

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

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. Alternatively, the chemiluminescent substrate is AMPPD.

It is noted that while the level of a biomarker in a 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 inventors have unexpectedly found that when combining a chemiluminescent method with the biomarkers Spondin1 and HE4 of the present application, at least the following advantages can be 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 radioimmunoassays and enzyme-linked immunoassays.

2. Has a wide linear power range. In the methods and kits of the present application, the luminous intensity is linear between 4-6 orders of magnitude and the concentration of the assay substance. This range is far greater than the linear range (2.0) of absorbance (OD value) in the enzyme immunoassay.

3. The result is stable, the error is small, the sample directly emits light, no light source is needed for irradiation, and the influence of a plurality of possible factors (light source stability, light scattering, light wave selector and the like) on analysis is eliminated, so that the analysis is sensitive, stable and reliable.

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

Examples

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

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

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

Magnetic particles: cargo number: magCOOH, available from SUZHIYI microsphere technology Co., ltd, under the trade name of carboxyl magnetic beads.

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

Murine anti-human HE4 monoclonal antibody (coated antibody), cat# HE4-McAb1#, commercially available from Phpeng organism under the trade name HE 4.

Mouse anti-human HE4 monoclonal antibody (detection antibody), cat# HE4-McAb2#, commercially available from the Phpeng organism under the trade name HE 4.

HE4 protein standard: the product number HE4-Ag is available from the Phpeng organism under the trade name HE 4.

Alkaline phosphatase: cargo number: SP011401, commercially available from chinese medicine agents under the trade name alkaline phosphatase.

AMPPD: CAS No. 122341-56-4, 4-methoxy-4- (3-phosphorylphenyl) spiro [1, 2-dioxycyclohexane-3, 2' -adamantane ], is an alkaline phosphatase substrate available from Beijing Gekko as a chemiluminescent substrate.

Tween-20: available from the organism under the trade name tween-20.

Example 1 establishment of a Spondin1 detection System and optimization thereof

And (3) constructing a detection system: coating carboxyl magnetic particles with 2.5 mug/mL of the mouse anti-human Spondin1 monoclonal antibody at 37 ℃ for 2 hours or coating magnetic particles at 4 ℃ overnight to prepare the magnetic particles coated by the mouse anti-human Spondin1 monoclonal antibody, namely a capture antibody; then, respectively adding the Spondin1 protein standard substances with the concentrations of 8000, 4000, 2000, 1000, 500 and 0pg/ml into different reaction cups, and reacting for 30 minutes at 37 ℃; then adding alkaline phosphatase-labeled murine anti-human Spondin1 monoclonal antibody (i.e., detection antibody) at a concentration of 0.25 μg/mL, reacting at 37 ℃ for 30 minutes, washing the magnetic beads, and removing the supernatant; finally, a luminescent substrate AMPPD is added, and the luminescence value is measured. Based on the luminescence values of the reaction system obtained from standard product values of different concentrations, a standard curve of the Spondin1 protein was constructed, and the results are 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 is more than or equal to 0.990 in the linear range, and the recovery rate is in the range of 90% -110%.

And (3) determining a detection system: antibodies of different concentrations were detected by checkerboard Fang Zhenfa, wherein the capture antibodies were selected from 0.5, 1.5, 2.5, 3.5, 4.5, 5.5 μg/mL, and the detection antibodies were selected from 0.05, 0.15, 0.2, 0.25, 0.35, 0.45, 0.55 μg/mL. The antibodies with different concentrations are detected, and the optimal working concentration of the Spondin1 capture antibody is found to be 2.5 mug/mL, and the optimal working concentration of the Spondin1 detection antibody is found to be 0.25 mug/mL.

Example 2 chemiluminescent detection kit for Spondin1

The specific components of the Spondin1 chemiluminescent detection kit constructed according to the Spondin1 serum detection system constructed in example 1 are shown in Table 1:

table 1. Components of Spondin1 chemiluminescent detection kit

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

Example 3 set up of HE4 serum detection reaction System and optimization thereof

Coating magnetic beads with a murine anti-human HE4 monoclonal antibody at a concentration of 5 μg/mL at 37℃for 2 hours or at 4℃overnight; then adding HE4 protein standard substance and serum sample with concentration of 0pmol/L, 20pmol/L, 40pmol/L, 80pmol/L, 160pmol/L, 320pmol/L, 640pmol/L into a sealing plate respectively, and reacting at 37 ℃ for 30 minutes; then, the mixture is reacted for 30 minutes at 37 ℃ by using a mouse anti-human HE4 monoclonal antibody marked by alkaline phosphatase with the concentration of 0.5 mug/mL, and the magnetic beads are washed to remove the supernatant; finally, a luminescent substrate AMPPD is added, and the luminescence value is measured. A standard curve of HE4 was constructed based on luminescence values of the reaction system obtained from standard product values of different concentrations, and the results are shown in FIG. 2. As shown in FIG. 2, the linear range of the HE4 chemiluminescent detection kit is 20pmol/L-640pmol/L, 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%.

And (3) determining a detection system: similar to the checkerboard method of example 2, the optimal working concentration of homemade HE4 capture antibody was found to be 5 μg/mL and the optimal working concentration of HE4 detection antibody was found to be 0.5 μg/mL by detecting antibodies at different concentrations.

The main components of the detection system were determined by the above study, and then the HE4 serum detection system was established.

Example 4 HE4 chemiluminescent detection kit

HE4 chemiluminescent detection kit was constructed according to the HE4 serum detection system set up in example 3, with the specific components shown in table 2 below:

table 2.HE4 chemiluminescent detection kit components

Evaluation of HE4 chemiluminescent detection kit: detecting HE4 repetitive reference by using HE4 chemiluminescence detection kit, wherein the detection is repeated for 10 times at the two levels of 40pmol/L and 80pmol/L of HE4 protein respectively, and the result shows that the variation coefficient CV is less than or equal to 10%; when the same sample is detected by 3 lot number kits, the variation coefficient CV between lots of the 3 lot number kits is less than or equal to 15%.

Example 5 human ovarian cancer marker Spondin 1-HE4 Joint detection kit

The Spondin1 chemiluminescent detection kit constructed in example 2 was combined with the HE4 chemiluminescent detection kit constructed in example 4 to form a Spondin 1-HE4 joint detection kit.

Example 6 Spondin 1-HE4 Joint detection kit diagnosis and prognosis of ovarian cancer

100 clinical samples were collected, 22 samples of the normal population, 78 samples of early stage ovarian cancer patients, and 1mL of serum per sample. Of the 78 ovarian cancer patients, 24 were stage I ovarian cancer patients and 54 were stage II ovarian cancer patients. Concentrations of the Spondin1 and HE4 markers in serum of ovarian cancer patients and healthy normal persons were detected, respectively, and ROC curves were drawn according to the detection results (fig. 3). The results showed that the cut-off value for Spondin1 to distinguish healthy and ovarian cancer patients was 2670pg/mL and the cut-off value for HE4 to distinguish healthy and ovarian cancer patients was 67.25pmol/L. The area under the curve, specificity and sensitivity of the Spondin1 and HE4 markers were then detected individually or in combination according to ROC curve statistics and the results are shown in table 3.

The results showed that the ovarian cancer group serum HE4 (591.96 + -384.5) U/mL and Spondin1 (3114.51 + -105.49) pg/mL, both were higher than the healthy control group HE4 (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 given in Table 3 below.

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

Marker(s)	Area under curve	Sensitivity of	Specificity (specificity)
				Spondin 1	0.782	74.3％	87％
HE4	0.854	77.1％	80％
				Spondin 1+HE4	0.921	87.1％	91.2％

Note that: the combined detection of the two markers of the Spondin1 and the HE4 is significantly 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 detecting the HE4 marker alone is 0.854, the sensitivity is 77.1%, and the specificity is 80%. The area under the curve when the two markers of Spondin1 and HE4 are combined to detect is 0.921, the sensitivity is 87.1%, and the specificity is 91.2%. It is clear that the combined detection of two markers, namely Spondin1 and HE4, is significantly superior to single ovarian cancer marker detection.

In conclusion, the Spondin1 and the HE4 can be used as markers for diagnosing and predicting early ovarian cancer, can be used for early diagnosis of ovarian cancer, and improves the accuracy of early prediction.

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

Claims (6)

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, evaluating or diagnosing ovarian cancer in the subject, the sample being blood, serum, plasma from the subject; the reagents for detecting the levels of Spondin1 and HE4 are antibodies to Spondin1 and to HE4, respectively; the antibodies to Spondin1 and to HE4 are a monoclonal antibody to Spondin1 and a monoclonal antibody to HE4, respectively; the optimal working concentration of the Spondin1 antibody is 0.25 μg/mL and the optimal working concentration of the HE4 antibody is 0.5 μg/mL.

2. The use according to claim 1, wherein the kit is a chemiluminescent kit comprising a capture antibody, a detection antibody and a chemiluminescent substrate.

3. The use according to claim 2, wherein the capture antibody is selected from the group consisting of a Spondin1 monoclonal antibody linked to magnetic particles, a HE4 monoclonal antibody linked to magnetic particles, and combinations thereof;

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; and/or the number of the groups of groups,

the chemiluminescent substrate is AMPPD.

4. A kit for predicting, evaluating or diagnosing ovarian cancer in a subject comprising reagents for detecting 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, the sample is blood, serum, plasma from the subject;

the reagents for detecting the levels of Spondin1 and HE4 are antibodies to Spondin1 and to HE4, respectively;

the antibodies to Spondin1 and to HE4 are a monoclonal antibody to Spondin1 and a monoclonal antibody to HE4, respectively.

5. The kit of claim 4, 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 magnetic particles, a HE4 monoclonal antibody linked to magnetic particles, and combinations thereof;

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; and/or the number of the groups of groups,

the chemiluminescent substrate is AMPPD.

6. The kit of claim 4, wherein the kit comprises 2 reagent subgroups,

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

Wherein subgroup 2 comprises: magnetic particles coated with a monoclonal antibody directed against HE4, HE4 monoclonal antibody with alkaline phosphatase label, and chemiluminescent substrate AMPPD.