CN101852805A - Application of ANGPTL3 as diagnostic marker of ovarian cancer - Google Patents

Abstract

The invention provides a method for diagnosing ovarian cancer in a tested person, which comprises the following steps of: detecting the content of ANGPTL3 in a sample from the tested person, analyzing the ANGPTL3 content and determining whether the tested person suffers from the ovarian cancer or not according to an analysis result. The invention also provides a reagent kit for ovarian cancer diagnosis.

Description

ANGPTL3 is as the purposes of the diagnostic marker of oophoroma

Technical field

The invention belongs to field of biomedicine technology.Specifically, the present invention relates to a kind of method and a kind of kit that is used for detecting sample ANGPTL3 albumen that improves sample classification accuracy.

Background technology

Epithelial ovarian cancer (epithelial ovarian cancer, EOC) position of in all women's cancer mortality reasons, being number five.The incidence of disease shared percent in women's common cancer in China's malignant tumor of ovary is 2.4-5.6%.Its morbidity is only second to cervical carcinoma in gynecologic malignant tumor, occupy second.Its morbidity is in rising trend in recent years.In female genital cancer knurl, oophoroma is the highest a kind of tumour of reason that causes death.ACS estimates that the U.S. will increase 21650 oophoroma cases in 2008 newly, and nearly 15520 routine sufferers will be died from this disease (www.cancer.org) simultaneously.The high mortality of EOC all is to occur in initial period, because most of women has been late period (stage III/IV) when diagnosis, wherein the patient of 15-20% has the survival rate (1) in 5 years.Comparatively speaking, accurately be diagnosed as the fraction patient of stage I, surpassing 90% has the survival rate (2) in 5 years.Candidate's strategy that current EOC detects is to be based upon on the basis of biological chemistry tumor markers, for example the biophysics label that obtains of CA125 and the doppler imaging by ultrasonic or ovary.Unfortunately, utilize these strategies to carry out positive predictive value (PPV) consistance of early stage EOC detection less than 10% (3,4).By utilize CA125 complicated algorithm (complex longitudinal algorithms) (5-7), series connection test (sequential testing) (8,9) and the application of brand-new biomarker (10), in to epithelial ovarian cancer (EOC) early diagnosis, obtained certain raising.So, still be starved of the biomarker that continues in the new blood circulation system of development now oophoroma carried out early diagnosis.

Summary of the invention

The present invention is based in part on following discovery: the ANGPTL3 content in the ovarian cancer patients serum is starkly lower than the ANGPTL3 content in health volunteer's serum, and has significance,statistical, can be used as the diagnosis marker of oophoroma.

Therefore, the invention provides following embodiment.

[embodiment 1] a kind of method of diagnosing the oophoroma among the experimenter comprises:

Detection is from the ANGPTL3 content in experimenter's the sample,

To described ANGPTL3 content analyze and

Determine according to analysis result whether described experimenter suffers from oophoroma.

[embodiment 2] a kind of method that improves sample classification accuracy comprises:

Detect the ANGPTL3 content in the described sample,

To described ANGPTL3 content analyze and

According to described analysis result, sample is classified.

[embodiment 3] a kind of oophoroma to the experimenter is diagnosed, the method for the monitoring of prognosis evaluation, result of treatment or course of disease monitoring, comprising: detect the expression of ANGPTL3 in experimenter's blood sample.

The method of each of [embodiment 4] embodiment 1-3, wherein said sample is selected from the group that is made of urine, cerebrospinal fluid, saliva and tears.

The method of each of [embodiment 5] embodiment 1-3, wherein said sample is a blood sample.

The method of each of [embodiment 6] embodiment 1-3, wherein said sample is a serum sample.

The method of each of [embodiment 7] embodiment 1-6, wherein said detection adopt ELISA or quantitative Western blot to carry out.

The method of each of [embodiment 8] embodiment 1-7, wherein said detection adopt the specific binding agents of ANGPTL3 to carry out.

The method of [embodiment 9] embodiment 8, the specific binding agents of wherein said ANGPTL3 are antibody or its ANGPTL3 binding fragment of ANGPTL3.

The method of [embodiment 10] embodiment 8, the antibody of wherein said ANGPTL3 is monoclonal antibody.

The method of [embodiment 11] embodiment 8, the antibody of wherein said ANGPTL3 is polyclonal antibody.

The method of each of [embodiment 12] embodiment 1-11, wherein said analysis comprises draws the ROC curve, is variable with ANGPTL3 wherein, goes out the ROC curve according to different threshold renderings, and area AUC under the calculated curve, and according to the expectation sensitivity and specificity judge.

The method of each of [embodiment 13] embodiment 1-11, wherein said analysis comprise with described ANGPTL3 content with compare from health volunteer's reference value, if be starkly lower than described reference value then determine that described experimenter suffers from oophoroma.

[embodiment 14] is used to implement each the kit of method of embodiment 1-13, and it comprises: the specific binding agents of ANGPTL3.

The kit of [embodiment 15] embodiment 14 also comprises: can be in conjunction with the labelled antibody of ANGPTL3.

The kit of each of [embodiment 16] embodiment 14-15, the specific binding agents of wherein said ANGPTL3 are antibody or its ANGPTL3 binding fragment of ANGPTL3.

The kit of [embodiment 17] embodiment 16, the antibody of wherein said ANGPTL3 is monoclonal antibody.

The kit of [embodiment 18] embodiment 16, the antibody of wherein said ANGPTL3 is polyclonal antibody.

The kit of each of [embodiment 19] embodiment 15-18, wherein said labelled antibody is by horseradish peroxidase or fluorescence labeling.

The kit of each of [embodiment 20] embodiment 14-19 also comprises:

The standard sample that comprises known quantity ANGPTL3 solution; And

The antibody labeling thing that is used to detect, it can form conjugate with antibodies.

The kit of [embodiment 21] embodiment 20, wherein said antibody labeling thing is horseradish peroxidase or fluorescent material.

The kit of each of [embodiment 22] embodiment 14-21 also comprises:

Optional antibody coupling matter (antibody conjugates);

The optional auxiliary reagent that is selected from the group that constitutes by developer, enzyme inhibitor, damping fluid, stabilizing agent, thinning agent, washing reagent; With

Optional instructions.

The kit of each of [embodiment 23] embodiment 14-22, the specific binding agents of wherein said ANGPTL3 is fixed on the solid phase carrier.

The specific binding agents of [embodiment 24] ANGPTL3 is used for the purposes of the reagent of diagnosis of ovarian cancer in preparation.

The purposes of [embodiment 25] embodiment 24, the specific binding agents of wherein said ANGPTL3 are antibody or its ANGPTL3 binding fragment of ANGPTL3.

The purposes of [embodiment 26] embodiment 25, the antibody of wherein said ANGPTL3 is monoclonal antibody.

The purposes of [embodiment 27] embodiment 25, the antibody of wherein said ANGPTL3 is polyclonal antibody.

[embodiment 28] ANGPTL3 is as the purposes of ovarian cancer diagnosis mark.

[embodiment 29] a kind of method that improves sample classification accuracy, it comprises:

Measure the content of ANGPTL3 in the sample and the content variation in time of ANGPTL3;

With ANGPTL3 content in time be changed to variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis are drawn out the ROC curve, and area AUC under the calculated curve;

According to AUC value, sensitivity and specificity sample is classified.

[embodiment 30] a kind of method that improves sample classification accuracy, it comprises:

Measure ANGPTL3 in the sample and arbitrary in oophoroma the content of high expressed albumen, and their variations in time of content separately;

With the ratio of ANGPTL3 and this high expressed protein content in time be changed to variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis are drawn out the ROC curve, and area AUC under the calculated curve;

According to AUC value, sensitivity and specificity sample is classified.

Studies show that, adopt method of the present invention and kit that albumin A NGPTL3 in the sample of individuality is carried out joint-detection, sensitivity, specificity and the accuracy rate of cancer diagnosis can be effectively improved, and diagnosis, prognosis evaluation, result of treatment assessment and the course of disease monitoring of numerous disease can be extended to.

Description of drawings

Fig. 1 is a width of cloth histogram, has shown the comparison of the expression of ANGPTL3 in healthy volunteer's serum (N1-N10) and ovarian cancer patients blood serum sample (C1-C10), and the T-check has significance,statistical (tail 1 for T-test, P=0.0009, and type 3).Horizontal ordinate is sample ID (a C=cancer, N=is normal); Ordinate is a concentration, and unit is every milliliter of nanogram (column diagram adds the error bar of standard error); Title is human angiogenin-like protein 3 (an oophoroma vs normal serum).

Embodiment

Term

Term used herein " ANGPTL3 " is meant PP1158 3 (angiopoietin-like protein 3 is called for short ANGPTL3), and its implication is well known in the art.ANGPTL3 is the member (11) of the angiopoietin-like family of secretory protein.It has the characteristic structure of angiogenin, comprises signal peptide, the terminal random coil domain of N-and the terminal fibrinogen (FBN) of C--spline structure territory.ANGPTL3 may in the adjusting of angiogenesis, play a role (11).ANGPTL3 is the instrumentality (12) of lipid metabolism, but its effect in cancer also is not studied.

Term used herein " experimenter " refers to any mammal, and for example, mouse, rat, rabbit, dog, ox, particularly primate are as the people.In some of the preferred embodiment of the invention, " experimenter " is the people.In this article, term " experimenter " and " individuality " are used interchangeably sometimes.

Term used herein " blood sample " refers to the sample from experimenter's blood, for example whole blood, blood plasma or serum sample.

Term used herein " specific binding agents of ANGPTL3 ", refer to can specific bond ANGPTL3 reagent, for example antibody of ANGPTL3, binding partner (as Aptmer) etc.

Term used herein " specific bond " refers to that specific binding agents selectivity of the present invention ground is in conjunction with ANGPTL3 antigen.For example, generally, the affinity of ANGPTL3 antibodies ANGPTL3 antigen is at least 2 times in conjunction with the affinity of the non-specific antigen outside the ANGPTL3 antigen (for example, BSA, casein).

" the ANGPTL3 binding fragment " of ANGPTL3 antibody is meant one or more fragments of ANGPTL3 antibody, and it keeps the ability in conjunction with ANGPTL3 antigen, for example Fab, F (ab ') ₂, Fv or strand Fv fragment.Shown the antigen combined function that to implement antibody by the fragment of full length antibody." the ANGPTL3 binding fragment " of antibody comprises (i) Fab fragment, and it is by V _L, V _H, C _LAnd C _H1The unit price fragment that domain is formed; (ii) F (ab ') ₂Fragment, it is to contain in the divalence fragment of hinge area by two Fab fragments of disulfide bond connection; (iii) by V _HAnd C _H1The Fd fragment that domain is formed; (iv) by the V of the single armed of antibody _LAnd V _HThe Fv fragment that domain is formed, (v) dAb fragment people such as (, (1989) Nature 341:544-546) Ward, it is by V _HDomain is formed; (the vi) complementary determining region of Fen Liing (CDR).In addition, although two domain VL of FV fragment and VH are by different gene codes, they can connect by synthetic joint through recombination method, and described joint makes VL and VH become a protein chain, and wherein VL and VH district pairing formation monovalent molecule (is called strand Fv (scFv); See, for example, people such as Bird (1988) Science242:423-426; With people (1988) Proc.Natl.Acad.Sci.USA 85:5879-5883 such as Huston).

Used term " monoclonal antibody " refers to the prepared product of the antibody molecule of single minute subconstiuent in the literary composition.The monoclonal antibody composition demonstrates single binding specificity and the compatibility to defined epitope.Therefore, term " monoclonal antibody " refers to demonstrate the antibody of single binding specificity.In one embodiment, produce human monoclonal antibodies by hybridoma, described hybridoma comprises from transgenic nonhuman animal, for example, obtain in the transgenic mice, with the B cell that immortalized cells merges, the genome of described animal comprises people's heavy chain transgenosis and light chain transgenosis.The preparation of antibody is well known in the art, and those skilled in the art can easily prepare the antibody of ANGPTL3.

Used term " labelled antibody " refers to be undertaken by labeled molecule the antibody of mark in the literary composition, for example by the antibody of marks such as fluorophore, chemiluminescent substance, horseradish peroxidase.(optional, optionally) expression " not essential " or implications such as " nonessential " " chosen " wantonly in term used herein.For example, " the optional instrument that carries " is meant to have this to carry instrument, also can not have this to carry instrument.This can according to circumstances be selected by those skilled in the art.

Kit

In a preferred embodiment, ELISA of the present invention uses kit to finish, and can realize prompt operation like this, thereby avoids the loaded down with trivial details of normal experiment detection.Preferred ELISA kit of the present invention comprises the ANGPTL3 detection kit.For improving sensitivity, specificity and the accuracy rate of medical diagnosis on disease, preferably use the ANGPTL3 detection kit, it can be respectively or records two groups of testing results simultaneously, to reach quick effect.A kind of particularly preferred ANGPTL3ELISA detection kit of the present invention is available from Immuno-BiologicalLaboratories Co.Ltd..

In a preferred embodiment, ANGPTL3 detection kit of the present invention comprises at least:

(1) can be in conjunction with the antibody of ANGPTL3; And

(2) when ANGPTL3 is incorporated into the antibody that limits in (1), can be incorporated into the labelled antibody of ANGPTL3.

The mentioned reagent box also can comprise:

(3) by the standard sample of the solution composition that contains known quantity ANGPTL3, this standard sample can derive from genetic engineering bacterium expression, animal or human's body fluid; And

(4) be used to the antibody labeling thing that detects, for example, as the enzyme label of method for reporting such as horseradish peroxidase, perhaps fluorescence labeling, it can form conjugate with antibodies.

More preferably, kit also can further comprise in the following article one of at least: (5) carry instrument, its spatial division is for accommodating the restriceted envelope of one or more containers, 96 orifice plates or lath etc., this container for example is medicine bottle, test tube and analog, and every sample container all can contain an independent component that is used for the inventive method; (6) auxiliary reagent, such as, developer, enzyme inhibitor, damping fluid, stabilizing agent, thinning agent, washing reagent and analog; (7) instructions, it can write on bottle, test tube and the analog, perhaps writes on the independent paper, perhaps outside or inner at container; Also can be multimedia form, such as CD, compact disk, video recording or the like.

Preferred antibody can be fixed on the solid phase carrier, forms capture antibody.Capture antibody is convenient especially in operation.

Contained antibody comprise any can be in conjunction with the antibody fragment of ANGPTL3, and can be recombinant, chimeric antibody, humanized antibody and mouse endogenous antibody.Described antibody can be monoclonal antibody or polyclonal antibody, preferred monoclonal antibody.

The enough ELISA reading machines of preferred antibody conjugate energy, such as, microplate reader is carried out photometering.

Sample

The used sample of the present invention can comprise various ways, such as whole blood, blood plasma, serum, urine, cerebrospinal fluid, saliva or tears.Wherein preferred serum.

The sample preparation can be carried out such as centrifugal grade according to commonsense method, for example, and referring to following document: Young, D.S.﹠amp; Bermes, E.W. " Specimen collection andprocessing " in Tietz Textbook of Clinical Chemistry 2nd Edition " Eds.Burtis, C.A.﹠amp; Ashwood, E.R., Saunders (1994); Methods inEnzymology, H.Van Vunakis and J.J.Langone (Eds), 1981,72 (B); Practice and Theory of Enzyme Immunoassays, P Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology, R.J.Burden and P.H.Van Knippenberg (Eds), Elsevier, 1985; Introduction toRadioimmunoassay and Related Techniques, T.Chard, ibid, 3rd Edition, 1987; Methods in Enzymology, H.Van Vunakis and J.J.Langone (Eds) 1981,74 (C).

Detection method

The present invention can use ELISA or other protein quantification technology the sample of individuality to be carried out the detection of albumin A NGPTL3.Preferably, can adopt the specific binding agents of ANGPTL3, for example the antibody of ANGPTL3 detects.In a preferred implementation, the antibody of ANGPTL3 is fixed on the solid phase carrier, for example uses as capture antibody.

(enzyme linked immunosorbentassay is a biology field protein content analyzing method commonly used ELISA) to enzyme-linked immunosorbent assay, and it can be used for measuring antigen, also can be used for measuring antibody.According to the source of reagent, the proterties of sample and the actual conditions of detection, the present invention can adopt number of different types, such as: double antibody sandwich method, dibit point single stage method, indirect method are surveyed the ELISA of antibody, competition law, prize law survey IgM antibody and application Avidin and biotin etc.Quantitatively Western blot also is a biology field protein content analyzing method commonly used.

Can also adopt quantitative test such as gelatin original position zymogram (gelatin zymography) or fluorescence method detection by the level of activity of chemical gauging ANGPTL3 enzyme.

The ROC curve

After detecting the concentration of ANGPTL3 in the sample with ELISA, available Mathematical Method is carried out statistical procedures to ANGPTL3 concentration in the sample that records, and obtains the grade scale with sample classification meaning on this basis.Such mathematical method is preferably finished by computing machine, such as drawing the ROC curve with these data, thereby the sample of individuality is classified.For example, individuality can be divided into cancer or health, therapeutic response is good and bad, and the prediction survival period is long and short etc.

ROC curve full name is experimenter's performance curve (receiver operatorcharacteristic curve), is called recipient's operating characteristic curve again, is mainly used in the clinical biochemical diagnostic test.The ROC curve be the reflection True Positive Rate (sensitivity claims susceptibility again, sensitivity) and false positive rate (1-specificity, the specificity) overall target of continuous variable are with announcement sensitivity of composition method and specific mutual relationship.It is by setting a series of different cut off value (threshold value or critical values, cut-off value, be to divide the normal and unusual dividing value of diagnostic test result) as continuous variable, thereby calculate a series of sensitivity and specificity, be that ordinate, 1-specificity are the horizontal ordinate curve plotting again with sensitivity, area under curve (AUC) is big more, and diagnostic accuracy is high more.On the ROC curve, the upper left point of the most close coordinate diagram is sensitivity and all higher critical value of specificity.ROC curve A UC value is between 1.0 and 0.5.Under the situation of AUC＞0.5, AUC approaches 1 more, illustrates that diagnosis effect is good more.AUC had low accuracy at 0.5～0.7 o'clock, AUC had certain accuracy at 0.7～0.9 o'clock, and AUC has high accuracy when above 0.9.

The evaluation method of ROC curve is different with traditional evaluation method, according to actual conditions, allows intermediateness, can be divided into a plurality of ordered categorizations to test findings, such as: normally, normal, suspicious, unusual and unusual five grades roughly roughly.

Above-mentioned ordered categorization for the diagnosis of disease, can be divided into: negative, uncertain, positive.Further, for cancer diagnosis, can be divided into: cancer, health.

In one embodiment, the present invention improves the method for sample classification accuracy, can comprise the steps:

(1) measures the content of albumin A NGPTL3 in the sample respectively;

(2) ratio with the ANGPTL3 protein content is variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis is drawn out the ROC curve, and area AUC under the calculated curve; And

(3) according to the sensitivity and the specificity of expectation, to measuring sample classify (cancer or health).

The drafting of ROC curve can be used software of the prior art or system, such as: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER_POWER.SAS, CREATE_ROC.SAS, GB STAT V10.0 (Dynamic Microsystems, Inc.Silver Spring, MD, USA) or the like.

The diagnosis of oophoroma, prognosis evaluation, result of treatment monitoring or course of disease monitoring

The present invention also provides a kind of joint test kit that is used for diagnosis, prognosis evaluation, result of treatment monitoring or the course of disease monitoring of oophoroma, and it comprises at least: (1) can be in conjunction with the antibody of ANGPTL3; And (2) can be incorporated into the labelled antibody of ANGPTL3 when ANGPTL3 is incorporated into the antibody that limits in (1).

In a preferred implementation, the invention provides that a kind of oophoroma to the experimenter is diagnosed, the method for prognosis evaluation, result of treatment monitoring or course of disease monitoring, comprise: in time variation of the content that detects the expression of ANGPTL3 in experimenter's blood sample and ANGPTL3 (as, various cancers developing period, before the treatment and treatment back etc.).The present invention also provides a kind of method that improves sample classification accuracy, and it comprises: in time the variation of the content of measuring the content of ANGPTL3 in the sample and ANGPTL3 (as, various cancers developing period, before the treatment and treatment back etc.); With the ANGPTL3 protein content in time be changed to variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis are drawn out the ROC curve, and area AUC under the calculated curve; According to AUC value, sensitivity and specificity sample is classified.For example, individuality can be divided into cancer or health, therapeutic response is good and bad, and the prediction survival period is long and short etc.In a preferred implementation, for example, can with ANGPTL3 content with compare from health volunteer's reference value, if be starkly lower than described reference value then determine that described experimenter suffers from oophoroma.In another preferred implementation, further combined with the low expression of ANGPTL3 and arbitrary in oophoroma the joint-detection of the albumen of high expressed, calculate the difference of their ratio, and with ratio in time be changed to variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis are drawn out the ROC curve, and area AUC under the calculated curve, and then sample classified.

Term used herein " high expressed albumen in oophoroma " is meant that the expression of this albumen in ovarian cancer patients is significantly higher than the normal subjects.The example of " albumen of high expressed in oophoroma " comprising: CA125, Mesothelin (Hassan R, Remaley AT, Sampson ML, Zhang J, Cox DD, Pingpank J, Alexander R, Willingham M, Pastan I, Onda M:Detection and quantitation of serum mesothelin, a tumor markerfor patients with mesothelioma and ovarian cancer.Clin Cancer Res 2006,12 (2): 447-453.), HE4, M-CSF, osteopontin, kallikrein (s), and soluble EGF receptor (soluble EGF receptor, Bast RC, Jr., Badgwell D, Lu Z, Marquez R, Rosen D, Liu J, Baggerly KA, Atkinson EN, Skates S, ZhangZ et al:New tumor markers:CA125 and beyond.Int J Gynecol Cancer2005,15Suppl 3:274-281.).

Below in conjunction with specific embodiment, the invention will be further described.Should be appreciated that following examples only to be used to the present invention is described and be not used to limit scope of the present invention in any form.

Embodiment 1

Below with the serum of individuality be sample to carry out ovarian cancer diagnosis be example, the present invention is described in further detail.

The collection of sample

The serum of healthy women of (34-82 year scope) of collecting serum sample and age-matched before the art of ovarian cancer patients is with comparing.In 2 hours of blood are got in venipuncture,, and aliquot sample is stored in-80 ℃ until use by centrifugal collection serum.

Survey ANGPTL3 content

Elisa assay-used human angiogenin-like protein 3 (ANGPTL3) the ELISA kit (cat#27409) of Immuno-Biological Laboratories Co.Ltd. exploitation.Elisa assay can be according to manufacturer's instructions operation.Utilize the GB STAT statistical packages of DynamicMicrosystems company exploitation to analyze the ROC curve.

And use Bio-Rad 680 type microplate reader (U.S.) to carry out the ELISA operation, comprising:

1, take out the kit of stored under refrigeration, place, recovery temperature is to room temperature (20-25 ℃).Sample sum and Quality Control number that calculating will detect.Each sample needs an antigen coated hole.Each experiment all will be done positive control, negative control and calibration object.Definite micropore quantity that needs.When lath temperature during to room temperature, open the protective bag of lath, take out antigen coated micropore lath.The reagent strip that this experiment does not need to use should be put into the sack of this resealable, and sealing is left 2-8 ℃ again in.

2, the titer, contrast liquid and the serum sample that on microwell plate, add 20 μ L successively.This process must be finished in 30 minutes.

3, the liquid of catching that adds 100 μ L in each micropore is blown and beaten repeatedly in the hole with pipettor and to be carried out mixing.

4, build plate with microwell plate, at room temperature (18-25 ℃) carried out incubation 55-65 minute.

5, wash plate 3 times by following step is manual:

A, acutely shake and outwell liquid in the plate;

B, in the hole, fill it up with washing lotion.Guarantee the remaining bubble of Kong Zhongwu.

C, repeat first two steps rapid twice again.

D, shake and remove the washing lotion in the hole.Plate is overturn, on paper handkerchief, pat, so that can pat dry all washing lotions.Observe microwell plate, guarantee the washing lotion of noresidue.

6, in the bottle of hrp-antibody complex, add the regeneration damping fluid of 7mL, deposit under the room temperature stand-by.

7, according to same order the good multienzyme complex of dilution is added 100 μ L to microwell plate

8, build plate with microwell plate, at room temperature (18-28 ℃) carried out incubation 55-65 minute.

9, preparation substrate adds a slice substrate solid in substrate buffer solution, dissolving is rocked strongly after 30-60 minute, and solution is mixed fully.

10, wash plate according to A in the step 5 to the method for D.

11, with same speed and order substrate solution is added 100 μ L in every hole.

12, microwell plate at room temperature (18-28 ℃) carried out incubation 55-65 minute.

13, the stop buffer that in each hole, adds 100 μ L with same speed and order.Should beat microwell plate gently after having added stop buffer, guarantee the whole mixings of sample.

14, the detection wavelength of microplate reader is set at the 405nm place.Detect the OD value in each hole.Ying Zaijia carry out reading within 15 clocks of stop buffer.

15, analyze the result of ANGPTL3 with a linear calibration curve " Y=mx+b ".

16, read the concentration of ANGPTL3 in serum sample and the contrast liquid by typical curve.

The result

Through above-mentioned steps, the testing result that obtains is as shown in table 1.

Table 1.ANGPTL3 is at normal population

With the expression among the oophoroma patients serum

Sample concentration (ng/ml)

c1 25.06

c2 111.86

c3 14.21

c4 75.69

c5 343.33

c6 412.04

c7 64.84

c8 93.78

c9 28.68

c10 129.94

n1 419.28

n2 260.14

n3 549.48

n4 307.16

n5 415.66

n6 278.23

n7 701.38

n8 90.16

n9 357.79

n10 451.83

^*C represents the cancer sample, and N represents normal specimens

As can be seen from Table 1, the concentration of ANGPTL3 and oophoroma symptom have close correlativity, and promptly the content of ANGPTL3 albumen is starkly lower than content in healthy volunteer's serum in the ovarian cancer patients serum.

We find that compare with described 10 normal individuals, the expression that described 10 oophoroma serum sample medium vesselses generate plain sample albumen 3 (ANGPTL3) lower (tail 1 for T-test, P=0.0009, and type 3) (Fig. 1).

The result shows that the concentration of ANGPTL3 and oophoroma symptom have close correlativity,

Related herein list of references comprises patent document, scientific paper, publication etc., all by reference its full content is included in herein.

Should be appreciated that under situation without departing from the spirit and scope of the present invention those of ordinary skill in the art can make various changes and improvements to it in form and details, and these all are considered to fall into protection scope of the present invention.

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Claims (30)

1. method of diagnosing the oophoroma among the experimenter comprises:

Detection is from the ANGPTL3 content in experimenter's the sample,

To described ANGPTL3 content analyze and

Determine according to analysis result whether described experimenter suffers from oophoroma.

2. method that improves sample classification accuracy comprises:

Detect the ANGPTL3 content in the described sample,

To described ANGPTL3 content analyze and

According to described analysis result, sample is classified.

Oophoroma to the experimenter diagnose, the method for prognosis evaluation, result of treatment monitoring or course of disease monitoring, comprising: detect the expression of ANGPTL3 in experimenter's blood sample.

4. the method for each of claim 1-3, wherein said sample is selected from the group that is made of urine, cerebrospinal fluid, saliva and tears.

5. the method for each of claim 1-3, wherein said sample is a blood sample.

6. the method for each of claim 1-3, wherein said sample is a serum sample.

7. the method for each of claim 1-6, wherein said detection adopt ELISA or quantitatively Western blot carry out.

8. the method for each of claim 1-7, wherein said detection adopt the specific binding agents of ANGPTL3 to carry out.

9. the method for claim 8, the specific binding agents of wherein said ANGPTL3 is antibody or its ANGPTL3 binding fragment of ANGPTL3.

10. the method for claim 8, the antibody of wherein said ANGPTL3 is monoclonal antibody.

11. the method for claim 8, the antibody of wherein said ANGPTL3 are polyclonal antibody.

12. the method for each of claim 1-11, wherein said analysis comprises draws the ROC curve, is variable with ANGPTL3 wherein, goes out the ROC curve according to different threshold renderings, and area AUC under the calculated curve, and according to the expectation sensitivity and specificity judge.

13. the method for each of claim 1-11, wherein said analysis comprise with described ANGPTL3 content with compare from health volunteer's reference value, if be starkly lower than described reference value then determine that described experimenter suffers from oophoroma.

14. be used to implement each the kit of method of claim 1-13, it comprises: the specific binding agents of ANGPTL3.

15. the kit of claim 14 also comprises: can be in conjunction with the labelled antibody of ANGPTL3.

16. the kit of each of claim 14-15, the specific binding agents of wherein said ANGPTL3 are antibody or its ANGPTL3 binding fragment of ANGPTL3.

17. the kit of claim 16, the antibody of wherein said ANGPTL3 are monoclonal antibody.

18. the kit of claim 16, the antibody of wherein said ANGPTL3 are polyclonal antibody.

19. the kit of each of claim 15-18, wherein said labelled antibody is by horseradish peroxidase or fluorescence labeling.

20. the kit of each of claim 14-19 also comprises:

The standard sample that comprises known quantity ANGPTL3 solution; And

The antibody labeling thing that is used to detect, it can form conjugate with antibodies.

21. the kit of claim 20, wherein said antibody labeling thing is horseradish peroxidase or fluorescent material.

22. the kit of each of claim 14-21 also comprises:

Optional antibody coupling matter;

The optional auxiliary reagent that is selected from the group that constitutes by developer, enzyme inhibitor, damping fluid, stabilizing agent, thinning agent, washing reagent; With

Optional instructions.

23. the kit of each of claim 14-22, the specific binding agents of wherein said ANGPTL3 is fixed on the solid phase carrier.

24.ANGPTL3 specific binding agents be used for the purposes of the reagent of diagnosis of ovarian cancer in preparation.

25. the purposes of claim 24, the specific binding agents of wherein said ANGPTL3 are antibody or its ANGPTL3 binding fragment of ANGPTL3.

26. the purposes of claim 25, the antibody of wherein said ANGPTL3 are monoclonal antibody.

27. the purposes of claim 25, the antibody of wherein said ANGPTL3 are polyclonal antibody.

28.ANGPTL3 purposes as the ovarian cancer diagnosis mark.

29. a method that improves sample classification accuracy, it comprises:

Measure the content of ANGPTL3 in the sample and the content variation in time of ANGPTL3;

With ANGPTL3 content in time be changed to variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis are drawn out the ROC curve, and area AUC under the calculated curve;

According to AUC value, sensitivity and specificity sample is classified.

30. a method that improves sample classification accuracy, it comprises:

Measure ANGPTL3 in the sample and arbitrary in oophoroma the content of high expressed albumen, and their variations in time of content separately;

With the ratio of ANGPTL3 and this high expressed protein content in time be changed to variable, according to different threshold values the sensitivity and the specificity of cancer diagnosis are drawn out the ROC curve, and area AUC under the calculated curve;

According to AUC value, sensitivity and specificity sample is classified.

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