CN109342731A

CN109342731A - The kit of gynecological tumor marker HER-2 and HE4 is detected simultaneously

Info

Publication number: CN109342731A
Application number: CN201811496854.8A
Authority: CN
Inventors: 张艺; 范俊; 周彬; 张珏; 韩婷婷; 谢敏浩
Original assignee: Wuxi Jiangyuan Industry Co Jiangsu Institute Of Nuclear Medicine; Jiangsu Institute of Nuclear Medicine
Current assignee: Wuxi Jiangyuan Industry Co Jiangsu Institute Of Nuclear Medicine; Jiangsu Institute of Nuclear Medicine
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2019-02-15

Abstract

The invention belongs to immunoassay fields, and in particular to while the kit and purposes of gynecological tumor marker HER-2 and HE4 is detected, the kit includes: the reagent containing tracer-labelling HER-2 antibody and tracer-labelling HE4 antibody；The HE4 antibody of the above-mentioned HER-2 antibody and tracer-labelling for using tracer-labelling can be with high degree of specificity and gynecological tumor marker HER-2 or HE4 antigen binding, and then it can use the kit, two tumor markers of people HER-2 and HE4 can be detected using double labelling time-resolved fluorescence immunoassay method simultaneously, facilitate analysis while breast cancer, oophoroma and carcinoma of endometrium, to improve the efficiency of screening and clinical interpretation.Based on the kit once-through operation can get two as a result, thus, improve checkability, reduce individually examine caused by error, be conducive to the screening and diagnosis of gynecological tumor.

Description

The kit of gynecological tumor marker HER-2 and HE4 is detected simultaneously

Technical field

The invention belongs to immunoassay fields, and in particular to it is a kind of detect simultaneously gynecological tumor marker HER-2 and The kit and purposes of HE4.

Background technique

Cancer has become the significant problem for threatening public health.In recent years, compared with male, Chinese women tumor invasion Average annual 2.2% significant growth is presented in rate.Wherein, the breast cancer of women spy's hair, the disease incidence of oophoroma and uterine cancer and death Rate rises year by year, and breast cancer accounts for the 15% of women new cancer, and uterine cancer accounts for 3.6%, and oophoroma accounts for 3%, city disease incidence compared with Rural area is significantly raised.To cope with Chinese women's idiopathic tumor breast cancer, the high-incidence status of oophoroma and uterine cancer, early discovery, Early diagnosis and early treatment are one of the effective means for improving survival.Serologic detection based on tumor markers has fast Prompt effective and simple and easy significant advantage, has become cancer early screening, detection and the important means of follow-up.

Newfound tumor markers improve the targeting of tumour, accuracy, help to increase diagnosis efficiency.As A kind of novel tumor markers, people's epididymal proteins 4 (human epididymis protein 4, HE4) are in normal ovarian group It knits in epithelium and does not express, and the high expression in oophoroma, uterus cancerous tissue, in cancer beside organism, normal tissue and benign tumour The important indicator that different degrees of low expression level is ovary, uterine malignant tumour diagnosis and identifies.US National health research Institute is by the detection of HE4 as the good pernicious important means of screening pelvic mass.Compared with conventional screening indexes CA125, HE4 egg White 100% expression in ovarian epithelial carcinoma, in benign tumour patient, 8% is positive, and CA125 positive rate 29%；In uterus In endometriosis patients, the former has 3%, and the latter is but up to 67%, and false positive rate is significant.Therefore, HE4 Endometrial Carcinomas and Value in oophoroma screening is substantially better than traditional CA125.ErbB-2 (human epidermal Growth factor receptor 2, HER-2) in the tumours such as human breast cancer, overexpression is presented, and HER-2 is positive Recurrence rate, the rate of transform of person is above Breast Cancer Patients with Negative Axillary.Chinese Breast Cancer guide detection in 2014 is informed: correct detection and HER-2 expression is evaluated, it is most important to the clinical diagnosis, treatment and Index for diagnosis of breast cancer.

Currently, there is HER-2 immunohistochemical diagnosis kit in the country, as disclosed in Chinese patent 200410020546.X Breast cancer HER-2 immunohistochemical diagnosis kit；There are the enzyme linked immunological kit of HE4, such as Chinese patent It is a kind of for detecting the enzyme linked immunological kit of HE4 disclosed in 201410348798.9.Mentioned reagent box, which exists, to be only capable of detecting The defect of single gynecological tumor marker, the former is only for the detection of breast cancer, and the latter is only for oophoroma, uterine cancer development Detection, causes the clinical diagnosis, treatment and Index for diagnosis of people's gynecological tumor to need to spend the time more, low efficiency.Above-mentioned examination simultaneously Agent box sensitivity and specificity are not high, and repeatedly individually detection is easy to produce biggish error, then lead to testing result confidence level It is low.

Summary of the invention

Therefore, the technical problem to be solved in the present invention is that the kit of existing detection gynecological tumor marker is only capable of quantitatively Or sxemiquantitative test, and carry out HER-2 and HE4 detection and must be tested twice, so the present invention provides it is a kind of simultaneously Detect the kit of gynecological tumor marker HER-2 and HE4.

For this purpose, the present invention provides a kind of kits for detecting gynecological tumor marker HER-2 and HE4 simultaneously, comprising: contain There is the reagent of tracer-labelling HER-2 antibody and tracer-labelling HE4 antibody.

In the kit, including respective independent packaging: containing the first HER-2 antibody of tracer-labelling and tracer The reagent of the first HE4 antibody of substance markers, using the 2nd HER-2 antibody and the coated antibody coating plate of the 2nd HE4 antibody, use HER-2 and HE4 antigen is prepared into calibration object solution, reaction buffer, concentration board-washing liquid and/or the enhancement solution of series of concentrations.

In the kit, the tracer is the substance containing lanthanide series.

In the kit, the lanthanide series includes but is not limited to europium, samarium, terbium or dysprosium.

In the kit, the lanthanide series is europium and samarium.

In the kit, HER-2 antigen series of concentrations range is 0-1000ng/mL in calibration object solution, preferably , it is 0-500ng/mL；HE4 antigen series of concentrations range is 0-20000pmol/L in calibration object solution, it is preferred that is 0- 1000pmol/L；Preferably, reaction buffer contains the bovine serum albumin(BSA) that mass concentration is 0.1-1%, it is preferred that contains quality The bovine serum albumin(BSA) that concentration is 0.2%；Preferably, the concentration board-washing liquid contains the surface-active that volumetric concentration is 1-10% Agent, it is preferred that the surfactant for being 5% containing volumetric concentration；Preferably, the enhancement solution is to contain beta-diketon body, trioctylphosphine The solution of phosphine oxide and acetic acid.

In the kit, the first HER-2 antibody or the 2nd HER-2 antibody are by following any antigen polypeptide system It is standby to form, and it is different from the antigen polypeptide of the 2nd HER-2 antibody to prepare the first HER-2 antibody:

(1) antigen polypeptide is as shown in SEQ ID NO:1；Or

(2) antigen polypeptide is as shown in SEQ ID NO:2.

In the kit, the first HE4 antibody or the 2nd HE4 antibody by following any antigen polypeptide preparation and At, and it is different from the antigen polypeptide of the 2nd HE4 antibody to prepare the first HE4 antibody:

(1) antigen polypeptide is as shown in SEQ ID NO:3；Or

(2) antigen polypeptide is as shown in SEQ ID NO:4.

In the kit, the component of the seminal plasma fructose detection kit are as follows:

The reagent containing tracer-labelling HER-2 antibody and tracer-labelling HE4 antibody, 2mL；

The antibody coating plate of the HER-2 antibody and HE4 antibody；

6 × calibration object the solution, every bottle of 1mL；

The reaction buffer, 50mL；

The concentration board-washing liquid, 40mL；Or

The enhancement solution, 30mL.

The present invention provides the kits for detecting gynecological tumor marker HER-2 and HE4 simultaneously to diagnose or detect in vitro Purposes in gynecological tumor.Preferably, the gynecological tumor includes breast cancer, oophoroma and carcinoma of endometrium.

Technical solution of the present invention has the advantages that

1, the kit of detection gynecological tumor marker provided by the invention, including contain tracer-labelling HER-2 antibody With the reagent of tracer-labelling HE4 antibody；It is above-mentioned using the HER-2 antibody of tracer-labelling and the HE4 antibody of tracer-labelling Can distinguish high degree of specificity with gynecological tumor marker HER-2 and HE4 antigen binding, and then can use the reagent Box can detect two tumor markers of people HER-2 and HE4 using double labelling time-resolved fluorescence immunoassay method simultaneously, Facilitate inspection while breast cancer, oophoroma and carcinoma of endometrium, and then improves the efficiency of screening and clinical interpretation.It is based on The kit, once-through operation can get two as a result, thus, improve checkability, reduce individually examine caused by error, Be conducive to the screening and diagnosis of gynecological tumor.

2, the kit of detection gynecological tumor marker provided by the invention, the first HER-2 antibody or the 2nd HER-2 Antibody is prepared by following any antigen polypeptide, and prepare resisting for the first HER-2 antibody and the 2nd HER-2 antibody Former polypeptide is different: (1) antigen polypeptide is as shown in SEQ ID NO:1；Or (2) described antigen polypeptide such as SEQ ID NO:2 institute Show；SEQ ID NO:1 is selected from the 23-1255 amino acids sequence of HER-2, and SEQ ID NO:2 is selected from the 23-640 of HER-2 Amino acids sequence, above-mentioned two antigen polypeptide have good antigenicity, high activity and high specific, the HER- prepared by it 2 antibody be capable of high degree of specificity with gynecological tumor marker HER-2 antigen binding, specific binding capacity is significantly higher than general The binding ability of logical HER-2 monoclonal antibody and HER-2 antigen, applies it to the detection of gynecological tumor marker HER-2, Detection sensitivity, specificity can be improved, reduce background noise to the full extent, further increase result credibility.

3, detection gynecological tumor marker kit provided by the invention, the first HE4 antibody or the 2nd HE4 antibody by Following any antigen polypeptide is prepared, and it is different from the antigen polypeptide of the 2nd HE4 antibody to prepare the first HE4 antibody: (1) The antigen polypeptide is as shown in SEQ ID NO:3；Or (2) described antigen polypeptide is as shown in SEQ ID NO:4；SEQ ID NO:3 31-124 amino acids sequence selected from HE4,32-123 amino acids sequence of the SEQ ID NO:4 selected from HE4, above-mentioned two Polypeptide fragment has good antigenicity, high activity and high specific, can be with high degree of specificity by its HE4 antibody prepared With gynecological tumor marker HE4 antigen binding, specific binding capacity is significantly higher than common HE4 monoclonal antibody and HE4 Therefore the binding ability of antigen applies it to the detection of gynecological tumor marker HE4, detection sensitivity, special can be improved Property, background noise is reduced to the full extent, further improves result credibility.

Detailed description of the invention

It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor It puts, is also possible to obtain other drawings based on these drawings.

Fig. 1 is the standard curve of HER-2 and HE4 in experimental example 1；

Fig. 2 is the standard curve of HER-2 and HE4 in experimental example 2.

Specific embodiment

Reagent involved in following embodiments or instrument are commercial product, and the product of different manufacturers is in the technical effect simultaneously Significant difference will not be brought, wherein Eu³⁺-N₂[P- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium, Sm³⁺-N₂[P- isocyanide Acid-benzyl]-diethylenetriamine tetraacethyl sodium, it is purchased from U.S. PE company；

Test serum is provided by Jiang Yuan hospital of Jiangsu Province；

The commission of the amino acid sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 Shanghai Sangon Biotech Company's synthesis；

HER-2 antigen, HE4 antigen, Tween-20, Triton X-100 and BSA, are purchased from Sigma company；

Cross-linked agarose gel 6B column is purchased from U.S. GE company；

Time-resolved fluorescence immunoassay instrument, PE company of the U.S., model Auto DELFIA 1235.

The preparation of embodiment 1HER-2-T antibody and HE4-T antibody

Amino acid sequence shown in SEQ ID NO:1 and SEQ ID NO:2 is utilized respectively as HER-2-T-1 antigen and HER- 2-T-2 antigen-immunized animal, to prepare HER-2-T-1 the and HER-2-T-2 antibody of specificity using above-mentioned antigen.Similarly, Amino acid sequence shown in SEQ ID NO:3 and SEQ ID NO:4 is utilized respectively to exempt from as HE4-T-1 antigen and HE4-T-2 antigen Epidemic disease animal, to prepare HE4-T-1 the and HE4-T-2 antibody of specificity using above-mentioned antigen.HER-2-T-1 and HER-2-T-2 Referred to as HER-2-T-1/2, HE4-T-1 and HE4-T-2 are referred to as HE4-T-1/2.

1. immune animal prepares monoclonal antibody:

1.1. take above-mentioned HER-2-T-1 antigen, HER-2-T-2 antigen, HE4-T-1 antigen and HE4-T-2 antigen (immune It is former) be sufficiently mixed respectively with isometric Freund's complete adjuvant (being purchased from Sigma company) after, Balb/c mouse is immunized, 50 μ g are anti- Former/only, subcutaneous multi-point injection.Survey serum titer after 4 weeks, the mouse for selecting immunoreactivity good booster immunization again: take antigen with After isometric incomplete Freund's adjuvant (being purchased from Sigma company) is sufficiently mixed, 25 μ of antigen dose g/, subcutaneous multi-point injection, The number of booster immunization is 3 times；Bump is immune twice again, and extracting spleen cell and Sp2/0 myeloma cell are routinely square later Method is mediated with 50%PEG (MW4000) (being purchased from Central Plains chemical company) and is merged, and (is purchased from Sigma with HAT conditioned medium Company) selection culture.CO is put into after fusion₂37 DEG C after culture 9~11 days in incubator, appearance biggish cell clone hole in. Start to be screened with indirect ELISA within 11 days.4 time cloning cultures are carried out (i.e. using limiting dilution assay to the hole of the primary dcreening operation positive Make screening after a large amount of schizogamies of cell), later amplifying cells, freeze.

1.2. it prepares ascites: Balb/c mouse only being handled with norphytane (purchased from Sigma company) 0.5ml/, after a week abdomen Chamber is inoculated with hybridoma 2 × 10⁶A/only, ascites is collected after 10 days.

1.3. measuring antibody titer: utilizing the HER-2-T-1 of HER-2-T-1 antigen preparation with indirect ELISA method measurement The potency of monoclonal antibody, the potency of monoclonal antibody reaches 1:33000 or more as the result is shown.

Similarly, it measures and reaches 1:33000 using the potency of the HER-2-T-2 monoclonal antibody of HER-2-T-2 antigen preparation More than；Potency using the HE4-T-1 monoclonal antibody of HE4-T-1 antigen preparation reaches 1:33000 or more；Utilize HE4-T-2 The potency of the HE4-T-2 monoclonal antibody of antigen preparation reaches 1:33000 or more.

2. immune animal prepares polyclonal antibody:

2.1. select the New Zealand White Rabbit that three monthly ages, weight are about 2kg or so as immune animal.In fundamental immunity, By the HER-2-T-1 antigen of the above-mentioned preparation of 1-2mg, HER-2-T-2 antigen, HE4-T-1 antigen and HE4-T-2 antigen (immunogene) Mixed respectively with isometric Freund's complete adjuvant, it is fully emulsified after rabbit back carry out multiple spot subcutaneous injection.Added every 4 weeks It is strong immune primary, antigen and incomplete Freund's adjuvant it is fully emulsified after, be only subcutaneously injected in back multiple spot with 100 μ g/.Last adds Strong immune latter 10th day arteria carotis intubation takes blood, separates serum.

2.2. antibody titer is measured: more using the H ER-2-T-1 of HER-2-T-1 antigen preparation with indirect elisa method measurement The potency of clonal antibody, antibody titer reaches 1:30000 or more as the result is shown.

Similarly, it measures and reaches 1:30000 using the potency of the HER-2-T-2 polyclonal antibody of HER-2-T-2 antigen preparation More than；Potency using the HE4-T-1 polyclonal antibody of HE4-T-1 antigen preparation reaches 1:30000 or more；Utilize HE4-T-2 The potency of the HE4-T-2 polyclonal antibody of antigen preparation reaches 1:30000 or more.

3. isolating and purifying antibody: ascites/serum is close after ammonium sulfate precipitation, then through Protein G (being purchased from Sigma company) And purifying.

4. being lyophilized after antibody packing, cryo-conservation.

Embodiment 2: the specificity identification of people HER-2-T-1/2 antibody and HE4-T-1/2 antibody

It is detected with ELISA.Respectively with people HER-2, people HE4, carcinomebryonic antigen (CEA), Neuron specific enolase Enzyme (NSE) (being purchased from Sigma company) is detection antigen coat elisa plate, then to detect prepared HER-2-T-1/2 respectively mono- The specific reaction of clonal antibody and HE4-T-1/2 monoclonal antibody and antigen plate is made negative with normal BALB/c mouse serum Control, PBS liquid make blank control.

As a result: HER-2-T-1/2 monoclonal antibody is only reacted with HER-2 for positive (P/N > 2.1), HE4-T-1/2 Dan Ke It is positive (P/N > 2.1) that grand antibody, which is only reacted with HE4, and reacting with CEA, NSE is feminine gender, illustrates HER-2-T-1/ of the invention 2 monoclonal antibodies and HE4-T-1/2 monoclonal antibody have specificity.

Utilize the specificity of above method identification HER-2-T-1/2 polyclonal antibody and HE4-T-1/2 polyclonal antibody, knot It is positive (P/N > 2.1) that fruit, which is that HER-2-T-1/2 polyclonal antibody only react with HER-2, HE4-T-1/2 polyclonal antibody with HE4 reaction is positive (P/N > 2.1), and reacting with CEA, NSE is feminine gender, illustrates HER-2-T-1/2 Anti-TNF-α of the invention Body and HE4-T-1/2 polyclonal antibody have specificity.

Embodiment 3 detects the preparation of gynecological tumor marker HER-2 and HE4 kit simultaneously

Kit that is a kind of while detecting gynecological tumor marker HER-2 and HE4 is present embodiments provided, including respectively solely Vertical packaging: marking the reagent of HER-2-T-1 antibody and lanthanide series label HE4-T-1 antibody containing lanthanide series, use HER-2-T-2 antibody and the coated antibody coating plate of HE4-T-2 antibody are prepared into series of concentrations using HER-2 and HE4 antigen Calibration object solution, reaction buffer, concentration board-washing liquid and/or enhancement solution.

Lanthanide series marks the preparation of HER-2-T-1 antibody-solutions (Eu-HER-2-T): to 1mg HER-2-T-1 monoclonal 0.2mg europium labelled reagent (Eu is added in antibody³⁺-N₂[P- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium), after mixing in 30 DEG C magnetic agitation 20 hours.Next day chromatographs the crosslinked Ago-Gel 6B column of the mixture, receives according to the flow velocity of 1ml/ pipe Collect eluent, measures the Eu fluorescence intensity of every pipe, collect first Eu eluting peak, by the Eu-HER-2-T solution of acquisition according to body Product is diluted than 1:200.

The preparation of lanthanide series label HE4-T-1 antibody-solutions (Sm-HE4-T): 1mg HE4-T-1 monoclonal antibody is taken to add Enter the samarium labelled reagent (Sm of 0.2mg³⁺-N₂[P- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium), in 30 DEG C of magnetic agitations 20 hours.Later, by the crosslinked Ago-Gel 6B column chromatography chromatography of the mixture, elution is collected according to the flow velocity of 1ml/ pipe Liquid measures the Sm fluorescence intensity of every pipe, collects first Sm eluting peak, by the Sm-HE4-T solution of acquisition according to volume ratio 1: 100 dilutions.

Mark the reagent of HER-2-T-1 antibody and lanthanide series label HE4-T-1 antibody by above-mentioned system containing lanthanide series The HER-2-T-1 antibody-solutions (Eu-HER-2-T) of standby lanthanide series label and the HE4-T-1 antibody that lanthanide series marks are molten Liquid (Sm-HE4-T) is mixed in equal volume, is saved backup for -20 DEG C after packing.

The preparation of antibody coating plate: by HER-2-T-2 monoclonal antibody and HE4-T-2 monoclonal antibody with pH9.6's 0.05M carbonate buffer solution is diluted to 5 μ g/mL, and every 100 μ L of hole coating is overnight；Next day discards coating buffer, is added then at every hole The above-mentioned carbonate buffer solution that 150 μ L contain 2%BSA is closed 2 hours, is then discarded, and vacuum drains coating plate, -20 DEG C of preservations.

The preparation of HER-2 and HE4 antigen calibration object solution: calibration object is diluted with the buffer containing bovine serum albumin(BSA) HER-2 and HE4 antigen is made at series of concentrations.The calibration object solution is BSA containing 2g/L and 1g/L NaN₃50mmol/ LTris-HCl reaction buffer (pH7.8).HER-2 the and HE4 antigen is configured to be containing the HER-2 antigen concentration 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL and containing the HE4 antigen concentration be 0pmol/ L, the mixing calibration object solution of 10pmol/L, 50pmol/L, 100pmol/L, 200pmol/L, 1000pmol/L.

The concentration board-washing liquid surfactant of 1-10% containing volumetric concentration.In the present embodiment, the concentration is washed Plate liquid is the Tris containing 12.1g/L, the NaCl of 312.43g/L, the Tween-20 of volumetric concentration 5%, with salt acid for adjusting pH to 7.8 Buffer solution.

The enhancement solution is the solution containing beta-diketon body, trioctyl phosphine oxide and acetic acid.In the present embodiment, described Enhancement solution be 3.7 ‰ containing volumetric concentration glacial acetic acid, the sodium acetate of 0.5g/L, β-naphthoyltrifluoroacetone of 0.05g/L, The mixed solution of the trioctyl phosphine oxide of 0.03g/L and the Triton X-100 for being 1 ‰ with volumetric concentration.

The reaction buffer contains the bovine serum albumin(BSA) that mass concentration is 0.1-1%.In the present embodiment, described Reaction buffer is the Tris containing 6.06g/L, the NaCl of 8.8g/L, the BSA, volumetric concentration 1g/L that mass concentration is 0.2% NaN₃, with the buffer solution of salt acid for adjusting pH to 7.8.

The formula of the seminal plasma fructose detection kit are as follows:

The antibody coating plate of the HER-2 antibody and HE4 antibody；

6 × calibration object the solution, every bottle of 1mL；

The reaction buffer, 50mL；

The concentration board-washing liquid, 40mL；Or

The enhancement solution, 30mL.

Embodiment 4 detects the kit of gynecological tumor marker HER-2 and HE4 simultaneously

Kit that is a kind of while detecting gynecological tumor marker HER-2 and HE4 is present embodiments provided, including respectively solely Vertical packaging: marking the reagent of HER-2-T-2 antibody and lanthanide series label HE4-T-2 antibody containing lanthanide series, use HER-2-T-1 antibody and the coated antibody coating plate of HE4-T-1 antibody are prepared into series of concentrations using HER-2 and HE4 antigen Calibration object solution, reaction buffer, concentration board-washing liquid and/or enhancement solution.

Lanthanide series marks the preparation of HER-2-T-2 antibody-solutions (Eu-HER-2-T): to 1mg HER-2-T-2 monoclonal 0.2mg Eu labelled reagent (Eu is added in antibody³⁺-N₂[P- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium), after mixing In 30 DEG C magnetic agitation 20 hours.Next day chromatographs the crosslinked Ago-Gel 6B column of the mixture, according to the flow velocity of 1ml/ pipe Collect eluent, measure the Eu fluorescence intensity of every pipe, collect first Eu eluting peak, by the Eu-HER-2-T solution of acquisition according to Volume ratio 1:200 dilution.

The preparation of lanthanide series label HE4-T-2 antibody-solutions (Sm-HE4-T): 1mg HE4-T-2 monoclonal antibody is taken to add Enter the Sm labelled reagent (Sm of 0.2mg³⁺-N₂[P- isocyanic acid-benzyl]-diethylenetriamine tetraacethyl sodium), in 30 DEG C of magnetic agitations 20 hours.Later, by the crosslinked Ago-Gel 6B column chromatography chromatography of the mixture, elution is collected according to the flow velocity of 1ml/ pipe Liquid measures the Sm fluorescence intensity of every pipe, collects first Sm eluting peak, by the Sm-HE4-T solution of acquisition according to volume ratio 1: 100 dilutions.

Mark the reagent of HER-2-T-2 antibody and lanthanide series label HE4-T-2 antibody by above-mentioned system containing lanthanide series Standby lanthanide series label HER-2-T-2 antibody-solutions (Eu-HER-2-T solution) and lanthanide series label HE4-T-2 antibody are molten Liquid (Sm-HE4-T solution) is mixed in equal volume, is saved backup for -20 DEG C after packing.

The preparation of antibody coating plate: by HER-2-T-1 monoclonal antibody and HE4-T-1 monoclonal antibody with pH9.6's 0.05M carbonate buffer solution is diluted to 5 μ g/mL, and every 100 μ L of hole coating is overnight；Next day discards coating buffer, and 150 μ are added in every hole L contains above-mentioned buffer blind 2 hours of 2%BSA, then discards, and vacuum drains coating plate, -20 DEG C of preservations.

The preparation of HER-2 and HE4 antigen calibration object solution: calibration object is diluted with the buffer containing bovine serum albumin(BSA) HER-2 and HE4 antigen is prepared at series of concentrations.The calibration object solution is BSA containing 2g/L and 1g/L NaN₃50mmol/L Tris-HCl buffer (pH7.8).By HER-2 the and HE4 antigen be configured to containing the HER-2 antigen concentration be 0ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL and containing the HE4 antigen concentration be 0pmol/L, The mixing calibration object solution of 10pmol/L, 50pmol/L, 100pmol/L, 200pmol/L, 1000pmol/L.

The concentration board-washing liquid surfactant of 1-10% containing volumetric concentration.In the present embodiment, the concentration is washed Plate liquid are as follows: the NaCl of the Tris containing 12.1g/L, 312.43g/L, the Tween-20 of volumetric concentration 0.5%, extremely with salt acid for adjusting pH 7.8 buffer solution.

The formula of the seminal plasma fructose detection kit are as follows:

The HE4 antibody reagent of the HER-2 antibody and tracer-labelling containing tracer-labelling, 2mL；

The coating plate of the HER-2 antibody and HE4 antibody；

6 × calibration object the solution, every bottle of 1mL；

The reaction buffer, 50mL；

The concentration board-washing liquid, 40mL；Or

The enhancement solution, 30mL.

Comparative example 1

This comparative example 1 is substantially the same manner as Example 3, and difference is only that, by HER-2-T-1 antibody therein and HE4-T-1 Antibody replaces with the HER-2 antibody of Abcam company of the U.S., HE4 antibody, and the antibody includes monoclonal antibody and Anti-TNF-α Body.

Experimental example 1

Detect the content of gynecological tumor marker HER-2 and HE4 simultaneously using kit described in embodiment 3, including such as Lower step:

Sample preparation: serum sample is the venous blood collected with serum tube, and centrifuging and taking supernatant uses.

Board-washing liquid is made of 1 bottle of concentration board-washing liquid (40mL) plus distilled water to 1L mixing.

The calibration object solution and each 50 μ L of sample to be tested and 50 μ L reaction buffers are taken respectively, and 25 DEG C of reactions 1 are small When, it is washed 4 times with board-washing liquid；It adds 100 μ L and contains lanthanide series label HER-2-T-1 antibody and lanthanide series label HE4- The reagent (mixed liquor of Eu-HER-2-T and Sm-HE4-T antibody) of T-1 antibody, 25 DEG C are reacted 1 hour, wash 6 with board-washing liquid Secondary, 100 μ L enhancement solutions are added in every hole, and oscillation detects fluorescence signal value after five minutes, with time-resolved fluorescence immunoassay instrument device, The fluorescent value of the calibration object solution and sample to be tested is obtained respectively, and the testing result in calibration object solution see the table below 1,2, then Standard curve is drawn according to the corresponding fluorescent value of the calibration object solution of the concentration of the calibration object solution and the concentration, with Fluorescence signal value is ordinate, and the concentration of corresponding HER-2 antigen or HE4 antigen is abscissa, fitting mark in calibration object solution Directrix curve, HER-2 standard curve are shown in that Fig. 1-A, HE4 standard curve are shown in Fig. 1-B, then detect the sample to be tested glimmering Light value is respectively (24135,101195,435217) HER-2, and HE4 (1266,3571,18435) substitutes into the corresponding HER-2 Or in HE4 standard curve, be calculated HER-2 content in the sample to be tested be respectively 6.4ng/mL, 30.5ng/mL, The content of 139ng/mL, HE4 are respectively 22.7pmol/L, 83.8pmol/L, 387.2pmol/L.

The testing result of table 1HER-2 calibration object

The testing result of table 2HE4 calibration object

Experimental example 2

Using the kit of comparative example 1, according to woman in the sample to be tested in the method while test experience example 1 of experimental example 1 The content of section tumor markers HER-2 and HE4 detect fluorescence signal value with time-resolved fluorescence immunoassay instrument device, obtain respectively The fluorescent value of the calibration object solution and sample to be tested is obtained, the testing result in calibration object solution see the table below 3,4, then according to institute The concentration fluorescent value corresponding with its for stating calibration object solution draws standard curve, and using fluorescence signal value as ordinate, calibration object is molten The concentration of corresponding HER-2 antigen or HE4 antigen is abscissa in liquid, and fit standard curve, HER-2 standard curve is shown in Fig. 2-A, HE4 standard curve is shown in Fig. 2-B, then by the fluorescent value that the sample to be tested detects be respectively HER-2 (16228,37910, 165219), (783,2671,8435) HE4 substitute into the corresponding HER-2 or HE4 standard curve, be calculated it is described to HER-2 content is respectively 5.4ng/mL, 26.4ng/mL, 117.1ng/mL in sample, and the content of HE4 is respectively 15.9pmol/ L、74.1pmol/L、328.1pmol/L。

The testing result of table 3HER-2 calibration object

The testing result of table 4HE4 calibration object

Experimental example 3

Sample preparation: serum sample is the venous blood collected with serum tube, and centrifuging and taking supernatant uses (in experimental example 1 Sample is identical).

Using the method in HER-2 and HE4 Electrochemiluminescince (ECLI) (kit is purchased from Roche Holding Ag) and experimental example 1 The sample to be tested is detected, in triplicate, the correctness of kit of the present invention is verified, as a result see the table below 5.

5 testing result of table

Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or It changes.There is no necessity and possibility to exhaust all the enbodiments.And thus amplify out it is obvious variation or It changes still within the protection scope of the invention.

SEQUENCE LISTING

<110>Jiangsu Inst of Atomic Medical Sciences

<120>kit of gynecological tumor marker HER-2 and HE4 is detected simultaneously

<130> WXHA201800203

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 1233

<212> PRT

<213>artificial synthesized

<400> 1

Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser

1 5 10 15

Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln

20 25 30

Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser

35 40 45

Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile

50 55 60

Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val

65 70 75 80

Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp

85 90 95

Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro

100 105 110

Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys

115 120 125

Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr

130 135 140

Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr

145 150 155 160

Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met

165 170 175

Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser

180 185 190

Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro

195 200 205

Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly

210 215 220

Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly

225 230 235 240

Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr

245 250 255

Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser

260 265 270

Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser

275 280 285

Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp

290 295 300

Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys

305 310 315 320

Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser

325 330 335

Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu

340 345 350

Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala

355 360 365

Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile

370 375 380

Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu

385 390 395 400

Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn

405 410 415

Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly

420 425 430

Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His

435 440 445

Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe

450 455 460

Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp

465 470 475 480

Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly

485 490 495

His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe

500 505 510

Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu

515 520 525

Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu

530 535 540

Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp

545 550 555 560

Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala

565 570 575

Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp

580 585 590

Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys

595 600 605

Thr His Ser Cys Val Asp Leu Asp Asp Lys Gly Cys Pro Ala Glu Gln

610 615 620

Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser Ala Val Val Gly Ile Leu

625 630 635 640

Leu Val Val Val Leu Gly Val Val Phe Gly Ile Leu Ile Lys Arg Arg

645 650 655

Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg Arg Leu Leu Gln Glu Thr

660 665 670

Glu Leu Val Glu Pro Leu Thr Pro Ser Gly Ala Met Pro Asn Gln Ala

675 680 685

Gln Met Arg Ile Leu Lys Glu Thr Glu Leu Arg Lys Val Lys Val Leu

690 695 700

Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys Gly Ile Trp Ile Pro Asp

705 710 715 720

Gly Glu Asn Val Lys Ile Pro Val Ala Ile Lys Val Leu Arg Glu Asn

725 730 735

Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met

740 745 750

Ala Gly Val Gly Ser Pro Tyr Val Ser Arg Leu Leu Gly Ile Cys Leu

755 760 765

Thr Ser Thr Val Gln Leu Val Thr Gln Leu Met Pro Tyr Gly Cys Leu

770 775 780

Leu Asp His Val Arg Glu Asn Arg Gly Arg Leu Gly Ser Gln Asp Leu

785 790 795 800

Leu Asn Trp Cys Met Gln Ile Ala Lys Gly Met Ser Tyr Leu Glu Asp

805 810 815

Val Arg Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys

820 825 830

Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu

835 840 845

Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly Gly Lys Val Pro Ile

850 855 860

Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg Arg Phe Thr His Gln

865 870 875 880

Ser Asp Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe

885 890 895

Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala Arg Glu Ile Pro Asp Leu

900 905 910

Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp

915 920 925

Val Tyr Met Ile Met Val Lys Cys Trp Met Ile Asp Ser Glu Cys Arg

930 935 940

Pro Arg Phe Arg Glu Leu Val Ser Glu Phe Ser Arg Met Ala Arg Asp

945 950 955 960

Pro Gln Arg Phe Val Val Ile Gln Asn Glu Asp Leu Gly Pro Ala Ser

965 970 975

Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu Leu Glu Asp Asp Asp Met

980 985 990

Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu Val Pro Gln Gln Gly Phe

995 1000 1005

Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly Gly Met Val His His

1010 1015 1020

Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly Gly Asp Leu Thr

1025 1030 1035

Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg Ser Pro Leu

1040 1045 1050

Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly Asp Leu

1055 1060 1065

Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His Asp

1070 1075 1080

Pro Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu

1085 1090 1095

Pro Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro

1100 1105 1110

Gln Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro

1115 1120 1125

Ser Pro Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala

1130 1135 1140

Thr Leu Glu Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val

1145 1150 1155

Val Lys Asp Val Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu

1160 1165 1170

Tyr Leu Thr Pro Gln Gly Gly Ala Ala Pro Gln Pro His Pro Pro

1175 1180 1185

Pro Ala Phe Ser Pro Ala Phe Asp Asn Leu Tyr Tyr Trp Asp Gln

1190 1195 1200

Asp Pro Pro Glu Arg Gly Ala Pro Pro Ser Thr Phe Lys Gly Thr

1205 1210 1215

Pro Thr Ala Glu Asn Pro Glu Tyr Leu Gly Leu Asp Val Pro Val

1220 1225 1230

<210> 2

<211> 618

<212> PRT

<213>artificial synthesized

<400> 2

Thr Gln Val Cys Thr Gly Thr Asp Met Lys Leu Arg Leu Pro Ala Ser

1 5 10 15

Pro Glu Thr His Leu Asp Met Leu Arg His Leu Tyr Gln Gly Cys Gln

20 25 30

Val Val Gln Gly Asn Leu Glu Leu Thr Tyr Leu Pro Thr Asn Ala Ser

35 40 45

Leu Ser Phe Leu Gln Asp Ile Gln Glu Val Gln Gly Tyr Val Leu Ile

50 55 60

Ala His Asn Gln Val Arg Gln Val Pro Leu Gln Arg Leu Arg Ile Val

65 70 75 80

Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr Ala Leu Ala Val Leu Asp

85 90 95

Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro Val Thr Gly Ala Ser Pro

100 105 110

Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser Leu Thr Glu Ile Leu Lys

115 120 125

Gly Gly Val Leu Ile Gln Arg Asn Pro Gln Leu Cys Tyr Gln Asp Thr

130 135 140

Ile Leu Trp Lys Asp Ile Phe His Lys Asn Asn Gln Leu Ala Leu Thr

145 150 155 160

Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys His Pro Cys Ser Pro Met

165 170 175

Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser Ser Glu Asp Cys Gln Ser

180 185 190

Leu Thr Arg Thr Val Cys Ala Gly Gly Cys Ala Arg Cys Lys Gly Pro

195 200 205

Leu Pro Thr Asp Cys Cys His Glu Gln Cys Ala Ala Gly Cys Thr Gly

210 215 220

Pro Lys His Ser Asp Cys Leu Ala Cys Leu His Phe Asn His Ser Gly

225 230 235 240

Ile Cys Glu Leu His Cys Pro Ala Leu Val Thr Tyr Asn Thr Asp Thr

245 250 255

Phe Glu Ser Met Pro Asn Pro Glu Gly Arg Tyr Thr Phe Gly Ala Ser

260 265 270

Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu Ser Thr Asp Val Gly Ser

275 280 285

Cys Thr Leu Val Cys Pro Leu His Asn Gln Glu Val Thr Ala Glu Asp

290 295 300

Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys Pro Cys Ala Arg Val Cys

305 310 315 320

Tyr Gly Leu Gly Met Glu His Leu Arg Glu Val Arg Ala Val Thr Ser

325 330 335

Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys Lys Ile Phe Gly Ser Leu

340 345 350

Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala

355 360 365

Pro Leu Gln Pro Glu Gln Leu Gln Val Phe Glu Thr Leu Glu Glu Ile

370 375 380

Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro Asp Ser Leu Pro Asp Leu

385 390 395 400

Ser Val Phe Gln Asn Leu Gln Val Ile Arg Gly Arg Ile Leu His Asn

405 410 415

Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu Gly Ile Ser Trp Leu Gly

420 425 430

Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly Leu Ala Leu Ile His His

435 440 445

Asn Thr His Leu Cys Phe Val His Thr Val Pro Trp Asp Gln Leu Phe

450 455 460

Arg Asn Pro His Gln Ala Leu Leu His Thr Ala Asn Arg Pro Glu Asp

465 470 475 480

Glu Cys Val Gly Glu Gly Leu Ala Cys His Gln Leu Cys Ala Arg Gly

485 490 495

His Cys Trp Gly Pro Gly Pro Thr Gln Cys Val Asn Cys Ser Gln Phe

500 505 510

Leu Arg Gly Gln Glu Cys Val Glu Glu Cys Arg Val Leu Gln Gly Leu

515 520 525

Pro Arg Glu Tyr Val Asn Ala Arg His Cys Leu Pro Cys His Pro Glu

530 535 540

Cys Gln Pro Gln Asn Gly Ser Val Thr Cys Phe Gly Pro Glu Ala Asp

545 550 555 560

Gln Cys Val Ala Cys Ala His Tyr Lys Asp Pro Pro Phe Cys Val Ala

565 570 575

Arg Cys Pro Ser Gly Val Lys Pro Asp Leu Ser Tyr Met Pro Ile Trp

580 585 590

Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln Pro Cys Pro Ile Asn Cys

595 600 605

Thr His Ser Cys Val Asp Leu Asp Asp Lys

610 615

<210> 3

<211> 94

<212> PRT

<213>artificial synthesized

<400> 3

Glu Lys Thr Gly Val Cys Pro Glu Leu Gln Ala Asp Gln Asn Cys Thr

1 5 10 15

Gln Glu Cys Val Ser Asp Ser Glu Cys Ala Asp Asn Leu Lys Cys Cys

20 25 30

Ser Ala Gly Cys Ala Thr Phe Cys Ser Leu Pro Asn Asp Lys Glu Gly

35 40 45

Ser Cys Pro Gln Val Asn Ile Asn Phe Pro Gln Leu Gly Leu Cys Arg

50 55 60

Asp Gln Cys Gln Val Asp Ser Gln Cys Pro Gly Gln Met Lys Cys Cys

65 70 75 80

Arg Asn Gly Cys Gly Lys Val Ser Cys Val Thr Pro Asn Phe

85 90

<210> 4

<211> 92

<212> PRT

<213>artificial synthesized

<400> 4

Lys Thr Gly Val Cys Pro Glu Leu Gln Ala Asp Gln Asn Cys Thr Gln

1 5 10 15

Glu Cys Val Ser Asp Ser Glu Cys Ala Asp Asn Leu Lys Cys Cys Ser

20 25 30

Ala Gly Cys Ala Thr Phe Cys Ser Leu Pro Asn Asp Lys Glu Gly Ser

35 40 45

Cys Pro Gln Val Asn Ile Asn Phe Pro Gln Leu Gly Leu Cys Arg Asp

50 55 60

Gln Cys Gln Val Asp Ser Gln Cys Pro Gly Gln Met Lys Cys Cys Arg

65 70 75 80

Asn Gly Cys Gly Lys Val Ser Cys Val Thr Pro Asn

85 90

Claims

1. a kind of kit for detecting gynecological tumor marker HER-2 and HE4 simultaneously, it is characterised in that: contain tracer-labelling The reagent of HER-2 antibody and tracer-labelling HE4 antibody.

2. kit according to claim 1, which is characterized in that including respective independent packaging: containing tracer-labelling The reagent of the first HE4 antibody of first HER-2 antibody and tracer-labelling is coated with using the 2nd HER-2 antibody and the 2nd HE4 antibody Antibody coating plate, the calibration object solution, the reaction buffer, concentration board-washing that are prepared into using HER-2 and HE4 antigen series of concentrations Liquid and/or enhancement solution.

3. kit according to claim 1 or 2, which is characterized in that the tracer is the substance containing lanthanide series.

4. kit according to claim 3, which is characterized in that the lanthanide series include but is not limited to europium, samarium, terbium or Dysprosium.

5. kit according to claim 3, which is characterized in that the lanthanide series is europium and samarium.

6. kit according to claim 2, which is characterized in that HER-2 antigen series of concentrations range in calibration object solution For 0-1000ng/mL, it is preferred that be 0-500ng/mL；HE4 antigen series of concentrations range is 0- in calibration object solution 20000pmol/L, it is preferred that be 0-1000pmol/L；Preferably, reaction buffer contains the ox that mass concentration is 0.1-1% Seralbumin, it is preferred that the bovine serum albumin(BSA) for being 0.2% containing mass concentration；Preferably, the concentration board-washing liquid contains body The surfactant that product concentration is 1-10%, it is preferred that the surfactant for being 5% containing volumetric concentration；Preferably, the enhancing Liquid is the solution containing beta-diketon body, trioctyl phosphine oxide and acetic acid.

7. kit according to claim 1-6, which is characterized in that the first HER-2 antibody or second HER-2 antibody is prepared by following any antigen polypeptide, and it is anti-with the first HER-2 to prepare the 2nd HER-2 antibody The antigen polypeptide of body is different:

(1) antigen polypeptide is as shown in SEQ ID NO:1；Or

(2) antigen polypeptide is as shown in SEQ ID NO:2.

8. kit according to claim 1-7, which is characterized in that the first HE4 antibody or the 2nd HE4 are anti- Body is prepared by following any antigen polypeptide, and prepares the antigen polypeptide of the first HE4 antibody and the 2nd HE4 antibody not It is same:

(1) antigen polypeptide is as shown in SEQ ID NO:3；Or

(2) antigen polypeptide is as shown in SEQ ID NO:4.

9. kit according to claim 1-8, which is characterized in that the component of the seminal plasma fructose detection kit are as follows:

The antibody coating plate of the HER-2 antibody and HE4 antibody；

6 × calibration object the solution, every bottle of 1mL；

The reaction buffer, 50mL；

The concentration board-washing liquid, 40mL；Or

The enhancement solution, 30mL.

10. claim 1-9 is described in any item while detecting the kit of gynecological tumor marker HER-2 and HE4 in vitro Purposes in diagnosis or detection gynecological tumor.Preferably, the gynecological tumor includes breast cancer, oophoroma and carcinoma of endometrium.