CN101525614A - Human oophoroma tumor marker HE4 enzymoimmunoassay kit - Google Patents

Abstract

The invention discloses a human oophoroma tumor marker HE4 enzymoimmunoassay kit, a preparation method and an application thereof. A PCR overlapping method is adopted to fully manually synthesize an HE 4 gene by a synthesized HE4 gene prime; the gene is constructed into a Rho GEX-4T1 expression vector for protein expression; the expressed HE4 fusion protein is restricted by enzyme and purified to obtain an HE4 purified product which immunes animals to obtain a polyclonal antibody and a monoclonal antibody; a rabbit antihuman HE4 polyclonal antibody is used to wrap and close an ELISA plate; the rabbit polyclonal antibody is used as a capture antibody, rat antihuman HE4 monoclonal antibody labeled by horseradish peroxidase is used as a detection antibody so as to be assembled into a double-antibody sandwiched ELISA kit for detecting HE4. The kit has the advantages of high detection sensitivity and high specificity, is suitable for the early diagnosis of oophoroma and provides oophoroma patients for curative effect observation and prognosis judgment with an easy, convenient, rapid, economical and reliable detection method.

Description

The human oophoroma tumor marker HE 4 enzymoimmunoassay test kit

Technical field

The invention belongs to that biotechnology and disease detection diagnostic reagent method and technology field, particularly HE4 gene are synthetic, vector construction, protein expression and purifying, HE4 is used for ELISA test kit and method for making thereof.

Background technology

Ovarian cancer morbidity concealment, poor prognosis is one of the most fatal malignant tumour of women.If diagnosis of ovarian cancer will improve curative ratio greatly in early days, but ovarian cancer patients often reached an advanced stage when finding.The most responsive index of its recurrence of current diagnosis ovarian cancer and monitoring is to detect change of serum C A125.Have 80% can detect the rising of CA125 among the advanced ovarian cancer patient, and only have 50～60% in early days, and CA125 also can rise in some benign disease, cause some unnecessary operations, limit its clinical application greatly.So many scholars are devoted to seek more efficiently tumor marker, so that make a definite diagnosis ovarian cancer early.

(human epididymis protein 4 HE4) finds in people's epididymal cell people's epididymis secretory protein 4.1999, Schummer etc. found mRNA high expression level in ovarian cancer tissue of HE4, expressed or did not express and hang down in healthy tissues.2003, the content of discovery HE4 such as Hellstrom in the serum of ovarian cancer patients also had obviously than the normal people and increases.Thereby HE4 has obtained the more deep research of people as the tumor markers of a new ovarian cancer.People such as Hellstrom confirm, be under 100% the situation in specificity, the sensitivity that HE4 detects ovarian cancer is 60%, and the sensitivity of classical oophoroma tumor marker CA125 only is 13%, and the susceptibility of HE4 detection early ovarian cancer also obviously is better than CA125.For further confirming and expanding HE4, carry out in existing nearly ten clinical studyes of the U.S. and clinical trial in the early detection of ovarian cancer and the application prospect aspect the course of disease monitoring.

Summary of the invention

The purpose of this invention is to provide a kind of HE4 method for synthesizing gene, protein expression, synthetic simple, the product purity height, the ELISA test kit preparation method of human oophoroma tumor marker HE4 also is provided in addition, judges the detection method that a kind of simple and efficient, economic and reliable is provided for oophoroma early diagnosis, observation of curative effect and prognosis.

The technical solution adopted in the present invention is: the HE4 gene is synthetic: design and synthesize eight PCR primers according to the gene order of the people HE4 that announces among the Genbank and in conjunction with the intestinal bacteria preference codon, obtain the HE4 gene by overlapping PCR method.

Described HE4 protein expression: the HE4 gene clone that will synthesize acquisition is gone in the pGEX-4T1 carrier, at expression in escherichia coli and be purified into reorganization GST-HE4 albumen.Obtain the pure product of HE4 albumen through zymoplasm cutting and purifying.

The method for making of described test kit: purifying HE4 protein immunization new zealand rabbit, preparation and purifying polyclonal antibody, with purifying HE4 protein immunization mouse, obtain hybridoma cell strain by cytogamy, with this hybridoma immune mouse, preparation ascites, the purified back of antibody horseradish peroxidase-labeled in the ascites, as capture antibody, as detecting antibody, set up the test kit that detects people HE4 double-antibody sandwich elisa with polyclonal antibody with the enzyme labelling monoclonal antibody.

The present domestic HE4 detection kit production of still not having, the test kit that the present invention developed is expected to substitute the import like product, for country saves a large amount of foreign exchanges, the diagnostic detection technology of China's ovarian cancer is reached world-class levels.

This test kit has high stability, susceptibility and specificity, and is simple to operate, quick, is suitable for early stage, the quick diagnosis of ovarian cancer.It is cheap that external like product is compared cost, the cost performance height.

Description of drawings

Fig. 1 is preparation technology's schema of this test kit.

Fig. 2 is through the HE4 gene DNA fragment of overlapping PCR acquisition and the double digestion qualification result figure of recombinant vectors pGEX-4T1-HE4.Wherein, the PCR result of 1:HE4 gene; 2:Marker DL2000; 3:Marker DL2000; 4:pGEX-4T1-HE4 double digestion result.

Fig. 3 is the SDS-PAGE electrophoresis (12%) of GST-HE4 fusion rotein IPTG abduction delivering, and enzyme is cut back proteic purification result of HE4 and immunoblotting qualification result figure.Wherein, the 1:pGEX-4T1 empty carrier is induced the result; 2:pGEX-4T1-HE4 does not induce contrast; 3:pGEX-4T1-HE4 induces the result; 4: albumen Marker; 5: albumen Marker; The 6:GST-HE4 fusion protease cuts except that the purification result behind the GST; 7: albumen Marker; 8: enzyme cut and purifying after the proteic immunoblotting result of HE4.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are used to illustrate the present invention, limit the scope of the invention and be not used in.Under the prerequisite that does not deviate from technical solution of the present invention, any change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.

Structure, protein expression, purifying and the evaluation of embodiment 1:HE4 gene

1.HE4 the amplification of gene

According to the people HE4mRNA sequence (NM_006103) that is provided among the Genbank, remove 30 signal peptides of N-terminal, according to the intestinal bacteria preference codon, 8 primers have been synthesized in design, difference called after P1～P8, primer is synthetic by the precious biotech firm in Dalian.

P1～P8 nucleotide sequence is formed:

P1：tggttgtgctactttttgttctttacctaatgataaagaaggttcttgtc

P2：ctaattgaggaaaattaatattaacttgaggacaagaaccttctttatca

P3：tctgaatgtgctgataatttaaaatgttgttctgctggttgtgctacttt

P4：ttgagaatcaacttgacattgatcacgacataaacctaattgaggaaaat

P5：ctgatcaaaattgtactcaagaatgtgtttctgattctgaatgtgctgat

P6：caaccattacgacaacatttcatttgaccaggacattgagaatcaacttg

P7：cgggatccgaaaaaactggtgtttgtcctgaattacaagctgatcaaaattgta

P8：ccgctcgagttattaaaaattaggagtaacacaagaaactttaccacaaccattacgacaa

5 ' end at P7 and P8 primer is introduced BamH I and Xho I restriction enzyme site respectively, and overlapping PCR method total man worker synthesizes the HE4 gene.First round reaction is with P1 and P2 pairing, generate product P 12, second take turns PCR with P12 as template, P3 and P4 generate product P 34 as primer, third round PCR with P34 as template, P5 and P6 as primer generate product P 56, the four-wheel PCR with P56 as template, P7 and P8 generate the HE4 gene as primer.The agarose gel electrophoresis observations is carried out in the PCR reaction according to a conventional method.

The result: use overlapping PCR joining method, successfully obtained the HE4 gene, fragment length is consistent with expection.

The HE4 gene nucleotide series:

1 gaaaaaactggtgtttgtcctgaattacaagctgatcaaaattgtactcaagaatgtgtt

61 tctgattctgaatgtgctgataatttaaaatgttgttctgctggttgtgctactttttgt

121?ctttacctaatgataaagaaggttcttgtcctcaagttaatattaattttcctcaatta

181?gtttatgtcgtgatcaatgtcaagttgattctcaatgtcctggtcaaatgaaatgttgt

241?cgtaatggttgtggtaaagtttcttgtgttactcctaatttt 282

2.pGEX-4T1-HE4 the structure of expression vector

Carrier pGEX-4T1 and the HE4 gene fragment behind the sepharose purifying, handle with BamH I and Xho I double digestion, enzyme is cut product and is connected behind the sepharose purifying, obtains recombinant plasmid pGEX-4T1-HE4, recombinant plasmid is identified through double digestion, simultaneously by the order-checking of the precious biotech firm in Dalian.With recombinant plasmid pGEX-4T1-HE4 transformed into escherichia coli, obtain the recombinant clone bacterium colony of pGEX-4T1-HE4.

As a result, successfully obtained recombinant plasmid pGEX-4T1-HE4, enzyme is cut qualification result as shown in Figure 2.The machine analysis as calculated of the sequencing result of recombinant plasmid shows that the HE4 fragment and the implementation sequence of reorganization is in full accord.

3.GST-HE4 Expression of Fusion Protein

The recombinant clone bacterium colony of pGEX-4T1-HE4 is cultivated A in the LB substratum that contains 100 μ g/ml penbritins ₆₀₀When reaching 0.5 left and right sides, add IPTG and induce, the IPTG final concentration is 0.5mmol/L, and induction time is 3h, and inducing temperature is 37 ℃, takes a morsel sample through the 12%SDS-PAGE electrophoresis observation behind the collection thalline.

As a result, the recombinant clone bacterium abduction delivering product of pGEX-4T1-HE4 determines that through the 12%SDS-PAGE electrophoresis fusion protein molecule amount of expression is about 37kD (Fig. 3).With this fusion rotein called after GST-HE4.

4.GST-HE4 the analysis of expressing fusion protein form

4 ℃ on bacterium after inducing, the centrifugal 10min of 8000rpm collects thalline, and PBS washs once, the centrifugal 10min of 8000rpm, wet bacterium is resuspended with PBS, places ice bath, carrying out ultrasonic bacteria breaking, the centrifugal 20min of 12000rpm goes up cleer and peaceful precipitation and does not carry out the 12%SDS-PAGE electrophoresis.

As a result, the 12%SDS-PAGE electrophoresis result shows that the GST-HE4 fusion rotein is the endochylema solubility expression.

5.GST-HE4 the purifying of fusion rotein and quantitative

Bacterium after collection is induced, PBS washs once, and wet bacterium precipitation is resuspended with PBS, places ice bath, ultrasonication, the centrifugal 20min of 12000rpm, supernatant are protein sample to be purified.With GST-Sepharose ^TMFiller is loaded in the purification column, purification column is connected with Akta purifying instrument, with PBS damping fluid (the method preparation that provides to specifications) 5 column volumes of balance, protein sample to be purified is with sample on the 1.5ml/min speed, be washed till baseline with level pad behind the last sample,, collect protein peak with albumen elution buffer wash-out, elutriant is 4 ℃ of dialysed overnight in PBS, get sample 12%SDS-PAGE electrophoretic analysis purification effect.Purifying protein after the dialysis is measured protein concentration with the Bradford method.

As a result, the GST-HE4 fusion rotein behind the GST affinity purification, the SDS-PAGE electrophoresis showed, single band is arranged about molecular weight 37kD, conform to desired value, gel thin-layer scanning shows that purity of protein reaches more than 90%, and it is 2mg/ml that the Bradford method is measured protein concentration.

6.GST-HE4 the enzyme of fusion rotein is cut and purifying

Concentration by 10U/mg adds zymoplasm in the GST-HE4 of purifying fusion rotein, 22 ℃ of enzymes are cut 16h, through Glutathione Sepharose4B sepharose column purification, collect effluent liquid, effluent liquid is further purified removal zymoplasm wherein, obtain highly purified HE4 albumen, getting in a small amount, sample carries out electrophoretic analysis with 12%SDS-PAGE.

As a result, obtain highly purified HE4 albumen after enzyme is cut, about molecular weight 10kD (Fig. 2), it is 1mg/ml that the Bradford method is measured protein concentration.

7. enzyme is cut the proteic immunoblotting evaluation of back HE4

Carry out with reference to method described in " molecular cloning ".The electrotransfer condition is 100V electrophoresis 1h, confining liquid is the TBST that contains 5% skim-milk, one anti-be the monoclonal antibody of commercial mouse-anti people HE4, and two resist and are the sheep anti-mouse igg antibody of horseradish peroxidase-labeled, carry out immunoblotting assay by the ECL Color Appearance System.

As a result, the HE4 albumen after enzyme is cut can with the monoclonal antibody generation immune response (Fig. 3) of mouse-anti people HE4.

Embodiment 2: the anti-people HE4 of rabbit Polyclonal Antibody Preparation

1. animal immune

Get the HE4 albumen 500 μ l (about 500 μ g) behind the purifying, fully emulsified with the equivalent Freund's complete adjuvant, adopt back subcutaneous multiple spot immunization immunity New Zealand large ear rabbit, later on once every two all immunity, protein immunization dosage is 300 μ g, adjuvant is a Freund's incomplete adjuvant, omnidistance immunity altogether 4 times, seven Lepus ear edge vein exploitating bloods after the last immunity.After indirect ELISA method was measured antibody titer, serum was collected in the carotid artery bloodletting, and-20 ℃ frozen.

2. the purifying of the anti-people HE4 of rabbit polyclonal antibody and the evaluation of tiring

Adopt Protein G affinitive layer purification rabbit anteserum, serum dilutes by a certain percentage with binding buffer liquid (sodium phosphate of 20mM), wash 5～10 column volumes with binding buffer liquid behind the last sample, use elution buffer (the glycine pH 2.5～3.0 of 0.1M) wash-out afterwards, collect elution peak, the Tris-HCl (pH9.0) that adds the 1M of 100 μ l rapidly in the elutriant of every milliliter of collection is so that antibody is in neutral environment.Elutriant 4 ℃ of dialysed overnight in PBS of collecting.

Adopt coupling that agarose (the CNBr-actived sepharose of the proteic cyanogen bromide-activated of HE4 is arranged again through the anti-people HE4 of the rabbit of ProteinG purifying polyclonal antibody ^TM4B) column purification, indirect ELISA method detect antibody titer (envelope antigen is the HE4 albumen of purifying) before and after the purifying, and ultraviolet spectrophotometer is measured antibody concentration, antibody after 0.22 μ m membrane filtration degerming ,-70 ℃ of preservations.

As a result, indirect ELISA detects and shows that tiring before the serum purifying is 1: 128 ten thousand, and tiring behind the purifying is 1: 64 ten thousand, shows to have obtained the high anti-people HE4 of rabbit polyclonal antibody of tiring, and antibody concentration is 1mg/ml.

Embodiment 3: mouse-anti people HE4 MONOCLONAL ANTIBODIES SPECIFIC FOR

1. animal immune

The HE4 albumen 300 μ l (about 300 μ g) that get purifying are fully emulsified with the equivalent Freund's complete adjuvant, the subcutaneous multi-point injection BALB/c mouse in back, altogether immune 3 mouse, every mouse immune antigen dose is 100 μ g, every mouse adds the subcutaneous multi-point injection of the fully emulsified back part of equivalent Freund's incomplete adjuvant with 50 μ g antigen doses in two week backs, later on once every two all immunity, every mouse immune dosage is 50 μ g, without adjuvant, immunization route is an abdominal injection, from immunity for the second time, the 7th day tail vein after each immunity gathered mouse blood, and indirect ELISA method detects serum titer (envelope antigen is the HE4 albumen of purifying), tire and reach above back preparation fusion in 1: 50000, merged preceding 3 days, the tail vein injection booster immunization once, antigen dose 50 μ g.

2. the foundation of hybridoma cell strain

2.1 cytogamy

Immune mouse after the eyeball bloodletting, the aseptic spleen of winning mouse, separating Morr. cell is expected blue dyeing counting through platform, gets about 1 * 10 ⁸Individual splenocyte, about 3 * 10 ⁷Individual SP2/0 myeloma cell, in the aseptic centrifuge tube of 50ml, the centrifugal 5min of 1200rpm abandons supernatant with two kinds of cytomixis, flicks the pipe end, makes the even pasty state of the loose one-tenth of sedimentation cell.In 37 ℃ of water-baths, rotate centrifuge tube on the other hand gently, another hand at the uniform velocity slowly splashes into 1ml 50% PEG4000 with dropper in 1min, in 1min, at the uniform velocity splash into the 1mlRPMI-1640 serum free medium then successively, at the uniform velocity splash into 2ml RPMI-1640 serum free medium in the 2min, at the uniform velocity splash into 10ml RPMI-1640 serum free medium in the 5min.Cell suspension is abandoned supernatant in the centrifugal 8min of 800rpm, selects to cultivate resuspension and mixing with HAT.Cell suspension is added in the 96 porocyte culture plates, and 100 μ l/ holes in 37 ℃, contain in the cell culture incubator of 5%CO2 and cultivate.Change HT on the 10th day and select substratum, changed the RPMI-1640 perfect medium that contains 20% foetal calf serum on the 14th day.

As a result, the 7th day visible cell clone that merges after the cytogamy, fusion rate reaches more than 90%.

2.2 the screening of positive colony and cloning

2～3 weeks after the cytogamy, when hybridoma grows to hole floorage 1/5 when above, as detecting antigen, screen by indirect ELISA method with the HE4 albumen of purifying, filter out can with the hybridoma of HE4 albumen test (with negative serum A ₄₅₀Ratio be judged to the positive greater than 2.1), 3 times (the 1st time subclone is selected nutrient solution with HT with the continuous subclone of limiting dilution assay, use common RPMI-1640 perfect medium instead the 2nd and the 3rd time), screened in 7-10 days behind each subclone, until the positive porosity of monoclonal cell is 100% o'clock, and the picking mono-clonal carries out liquid nitrogen cryopreservation after the enlarged culturing.

As a result, obtaining 3 strains altogether can the proteic hybridoma cell strain of the anti-people HE4 of stably excreting, called after H1, B6 and F4 respectively.

2.3 the evaluation of monoclonal antibody hypotype

Use mouse monoclonal antibody hypotype identification kit (available from Hycult biotechnology company) and identify the monoclonal antibody hypotype, by operation instructions three strain hybridoma supernatants are used suitably dilution of PBS (pH 7.4) respectively, to antibody concentration 1-50 μ g/ml, add the diluent that provides in the 500 μ l test kits to the detector tube that contains a κ type test strip, add 500 μ l again and dilute good sample to be checked to detector tube, test strip is immersed and stirring gently fully, the RAM-kappa that provides in the 1ml test kit is provided in detector tube again, make the test strip printing surface down, about room temperature vibration 30min, observations.

As a result, the monoclonal antibody hypotype of three strain hybridoma supernatants all is accredited as the IgG1 class, and light chain subtype is the κ chain.

3. ascites prepares and purifying

Freund's incomplete adjuvant sensitization BALB/c mouse, 500 μ l/ only.Sensitization one all pneumoretroperitoneum injection hybridoma suspensions (are selected the H1 hybridoma cell strain, cell count 5 * 10 ⁶～1 * 10 ⁷), gather mouse ascites after 7～10 days, 12000rpm, centrifugal 5min collects supernatant.The silicon-dioxide absorption method is carried out pre-treatment to ascites, adopts sad-saturated ammonium sulphate method to carry out purifying: pretreated ascites and acetate buffer solution (0.06mol/L, pH 5.0) were mixed in 1: 2 by volume, with the HCl accent pH to 4.8 of 1mol/L; Add the sad ratio of 11 μ l in every milliliter of mixed solution, it is sad dropwise to add under the stirring at room, adds in 30min, 4 ℃ leave standstill 2h, the centrifugal 30min of 12000rpm, supernatant add the PBS of 1/10 volume after 125 μ m nylon mesh are filtered, with the NaOH accent pH to 7.2 of 1mol/L; Add saturated ammonium sulphate down at 4 ℃, leave standstill 1h; The centrifugal 30min of 12000rpm, abandon supernatant, precipitation is dissolved among an amount of PBS (containing 137mmol/L NaCl, 2.6mol/L KC1,0.2mmol/L EDTA), with 100 times of PBS with upper volume, 4 ℃ of dialysed overnight, the last centrifugal 30min of 12000rpm collects supernatant, ultraviolet spectrophotometer is measured monoclonal anti body burden behind the purifying, behind the 0.22 μ l membrane filtration in-70 ℃ of preservations.

As a result, ultraviolet spectrophotometer mensuration antibody concentration is 15mg/ml.

4. the indirect ELISA of mouse-anti people HE4 monoclonal antibody is identified behind the purifying

With the HE4 albumen of purifying as detecting antigen, with the mouse ascites doubling dilution behind the purifying, as negative control, use indirect ELISA method and detect with the normal mouse serum of dilution in 1: 200, with positive greater than 2.1 sections of declaring, calculate monoclonal antibody and tire with the ratio of negative serum.

As a result, tiring of the mouse ascites of purifying reaches 1: 256000.

5. the monoclonal antibody of horseradish peroxidase-labeled purifying

Obtain ELIAS secondary antibody with the periodates oxidation style: dissolving 5mg horseradish peroxidase is in the 1.2ml ultrapure water, the sodium periodate 0.3ml that adds the 0.1mol/L of new preparation, room temperature leaves standstill 20min, with above-mentioned solution 4 ℃ of dialysis 24h in the sodium-acetate of 1mmol/L, during every 4h change dialyzate.Sodium carbonate solution with 0.02mol/L prepares the monoclonal antibody sample to 10mg/ml, the antibody-solutions that 0.5ml is prepared adds in the horseradish peroxidase solution of dialysis, room temperature leaves standstill 2h, in mixed solution, add 100 μ l sodium borohydrides (aqueous solution of 4mg/ml), 4 ℃ of reaction 2h, in mixed solution, add isopyknic saturated ammonium sulphate, the centrifugal 15min of 12000rpm behind 4 ℃ of reaction 30min, abandon supernatant, precipitation is dissolved among the PBS of 1ml, dialysed overnight in PBS is got supernatant behind the centrifugal 15min of 12000rpm next day and is added isopyknic glycerine ,-70 ℃ of preservations after the packing.The ELIAS secondary antibody that takes a morsel, as envelope antigen, indirect ELISA method detects with the HE4 of purifying.

As a result, detect enzyme mark monoclonal antibody through indirect ELISA method and can under dilution in 1: 10000, obtain good detection effect.

Embodiment 4: the foundation of human oophoroma tumor marker HE4 double-antibody sandwich elisa detection method

1. detection method

In the present embodiment, the anti-people HE4 of the rabbit polyclonal antibody that obtains in the Application Example 2 is a capture antibody, with the mouse-anti people HE4 monoclonal antibody of the horseradish peroxidase-labeled that obtains among the embodiment 3 as detecting antibody, set up the how anti-and monoclonal antibody double-antibodies sandwich ELISA that detects oophoroma tumor marker HE4.

2. enzyme plate bag quilt

With coating buffer (carbonate buffer solution of 0.05mol/L, pH 9.6) the anti-people HE4 of rabbit polyclonal antibody is diluted to 10 μ g/ml, mixing, every hole adds 100 μ l and dilutes good how anti-solution in the 96 hole enzyme plates, enzyme plate is sealed in 4 ℃ spends the night.

3. enzyme plate sealing

With the PBS that contains 3% isinglass and 5% sucrose as confining liquid.The enzyme plate that spent the night of bag is patted dry, and every hole adds 200 μ l confining liquids, and 37 ℃ of sealing 1h pat dry enzyme plate after plate liquid (TBST) is washed plate 3 times with washing.Be encapsulated in the metallic foil bag that siccative is housed 4 ℃ of preservations after the enzyme plate drying.

4. the processing of tested serum

Tested serum is made the dilution of suitable proportion of sample diluting liquid (PBS that contains 1%BSA and 0.05% tween 20).100 μ l/ holes pat dry enzyme plate after plate liquid is washed plate 3 times with washing behind 37 ℃ of reaction 1h.

5. the processing of ELIAS secondary antibody

ELIAS secondary antibody is stored among the PBS that contains 50% glycerine, does 1: 10000 dilution back with the PBS of pH 7.2 and uses matching while using.100 μ l/ holes pat dry enzyme plate after plate liquid is washed plate 3 times with washing behind 37 ℃ of reaction 30min.

6. colour developing and termination

Develop the color with the TMB Color Appearance System.Colour developing liquid preparation: A liquid (citric acid of 0.05mol/L, the Sodium phosphate dibasic of 0.1mol/L) 6ml+B liquid (containing in the dehydrated alcohol of 2mg/ml TMB) 0.3ml+C liquid (0.75%H ₂O ₂) 20 μ l, matching while using, 50 μ l/ holes, 37 ℃ of lucifuges are reacted 15min, add the sulfuric acid of 2M afterwards, 50 μ l/ holes.

7. detected result

This experimental result is with the A of ELISA experiment ₄₅₀Value representation.

8. contrast

The test sample of negative control hole is the sample diluting liquid of increase serum not, and the good HE4 albumen of purifying is as the positive criteria product.

Embodiment 5: the composition of human oophoroma tumor marker HE4 double-antibody sandwich elisa test kit

1.ELISA plate: wrapped by the anti-people HE4 of the rabbit of good purifying polyclonal antibody and also sealed.

2. sample diluting liquid to be checked: the PBS that contains 1%BSA and 0.05% tween 20.

3. ELIAS secondary antibody: the mouse-anti people HE4 monoclonal antibody of horseradish peroxidase-labeled is stored in the PBS that contains 50% glycerine.

4. the PBS of ELIAS secondary antibody diluent: pH 7.2

5.10 * wash the Tris-HCl of plate liquid: 0.2mol/L, the NaCl of 1.5mol/L, 0.5% tween 20

6. colour developing liquid: A liquid is the citric acid of 0.05mol/L and the Sodium phosphate dibasic of 0.1mol/L, and B liquid is the dehydrated alcohol that contains 2mg/ml TMB, and C liquid is 0.75%H ₂O ₂

7. the sulfuric acid of stop buffer: 2M.

8. positive criteria product: the HE4 albumen of purifying.

Embodiment 6: the experimental performance of human oophoroma tumor marker HE4 double-antibody sandwich elisa test kit

1. between the detection zone of test kit

Get the enzyme plate that bag has also been carried out sealing by the anti-people HE4 of good rabbit polyclonal antibody, each sample is done three multiple holes, adds the HE4 protein standard substance with the sample diluting liquid gradient dilution, 100 μ l/ holes, 37 ℃ of reaction 1h wash plate liquid and wash plate 3 times, enzyme plate is patted dry, add the ELIAS secondary antibody of dilution in 1: 10000,100 μ l/ holes, 37 ℃ of reaction 30min wash plate liquid and wash plate 3 times, and enzyme plate is patted dry, add colour developing liquid 50 μ l/ holes, 37 ℃ of lucifuges are reacted 15min, add the sulfuric acid of 2M afterwards, 50 μ l/ holes.Microplate reader detects A ₄₅₀, be X-coordinate with the HE4 standard substance albumen of different concns, with corresponding A ₄₅₀Value is ordinate zou, drawing standard curve.

As a result, this test kit has between the detection zone of good linear relation and is 15pM～900pM, if the HE4 concentration of tested serum is higher than time useful range, detects after then should using the sample diluting liquid dilute sample again.

2. the precision of test kit

Adopt 4 parts of human serum samples to test, the multiple hole of every part of sample is detected, and carry out 2 times detect every day in the different time in 20 days.Total CV value＜10% of this detection kit precision, the data results of this research is as follows:

3. the detectability of test kit

What detectability representative was different from zero-dose can the antigenic minimum concentration of detected HE4.Adopt not contain the proteic sample diluting liquid of HE4 and 4 parts of serum that derive from healthy people, the latter is diluted to 5pM with sample diluting liquid, carries out 4 times and detect in two Independence Days, repeats 24 times at every turn and detects.

As a result, the detectability＜5pM of this test kit.

4. the rate of recovery of test kit

The HE4 albumen of concentration known is joined in the normal human serum sample, sample is diluted, measure the concentration of HE4.HE4 concentration (pM)/(the HE4 concentration (pM) of endogenous HE4 concentration (pM)+adding) * 100% of the % rate of recovery=record.The rate of recovery of calculating this test kit is (100 ± 15) %, and the data results of this research is as follows:

5. the application example of test kit

Use this test kit to detect the content of HE4 in 65 routine women's serum altogether, healthy person 20 examples wherein, before the art through being diagnosed as ovarian cancer patients 45 examples.In this research, the HE4 detected value of 95% healthy women individuality is positioned at or is lower than 150pM, and in the ovarian cancer patients serum, HE4 content significantly raises, 95.6% reaches more than the 300pM, and this detected result has the very consistence of high level with clinical data, conforms to the report of domestic and international pertinent literature.

SEQUENCE?LISTING

＜110〉Dalian Meiyide Biology Technology Co., Ltd.

＜120〉human oophoroma tumor marker HE 4 enzymoimmunoassay test kit

<130>A?novel?multiple?marker?bioassay?utilizing?HE4?and?CA125?for?the

prediction?of?ovarian?cancerin?patients?with?a?pelvic?mass

<160>10

<170>PatentIn?version?3.5

<210>1

<211>50

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P1

<400>1

tggttgtgct?actttttgtt?ctttacctaa?tgataaagaa?ggttcttgtc 50

<210>2

<211>50

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P2

<400>2

ctaattgagg?aaaattaata?ttaacttgag?gacaagaacc?ttctttatca 50

<210>3

<211>50

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P3

<400>3

tctgaatgtg?ctgataattt?aaaatgttgt?tctgctggtt?gtgctacttt 50

<210>4

<211>50

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P4

<400>4

ttgagaatca?acttgacatt?gatcacgaca?taaacctaat?tgaggaaaat 50

<210>5

<211>50

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P5

<400>5

ctgatcaaaa?ttgtactcaa?gaatgtgttt?ctgattctga?atgtgctgat 50

<210>6

<211>50

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P6

<400>6

caaccattac?gacaacattt?catttgacca?ggacattgag?aatcaacttg 50

<210>7

<211>54

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P7

<400>7

cgggatccga?aaaaactggt?gtttgtcctg?aattacaagc?tgatcaaaat?tgta 54

<210>8

<211>61

<212>DNA

<213>Artificial?Sequence

<220>

＜223〉according to intestinal bacteria preference codon synthetic HE4 primer P8

<400>8

ccgctcgagt?tattaaaaat?taggagtaac?acaagaaact?ttaccacaac?cattacgaca 60

a 61

<210>9

<211>282

<212>DNA

<213>Artificial?Sequence

<220>

＜223 〉, adopt the HE4 gene order of overlapping PCR method splicing according to behind the intestinal bacteria preference codon synthetic primer

<400>9

gaaaaaactg?gtgtttgtcc?tgaattacaa?gctgatcaaa?attgtactca?agaatgtgtt 60

tctgattctg?aatgtgctga?taatttaaaa?tgttgttctg?ctggttgtgc?tactttttgt 120

tctttaccta?atgataaaga?aggttcttgt?cctcaagtta?atattaattt?tcctcaatta 180

ggtttatgtc?gtgatcaatg?tcaagttgat?tctcaatgtc?ctggtcaaat?gaaatgttgt 240

cgtaatggtt?gtggtaaagt?ttcttgtgtt?actcctaatt?tt 282

<210>10

<211>94

<212>PRT

<213>human

<300>

<301>Hellstrom，I.，Raycraft，J.，Hayden-Ledbetter，M.，Ledbetter，J.A.，

Schummer，M.，McIntosh，M.，Drescher，C.，Urban，N.and

Hellstrom，K.E.

<302>The?HE4(WFDC2)protein?is?a?biomarker?for?ovarian?carcinoma

<303>Cancer?Res.63(13)，3695-3700(2003)

<304>63

<305>13

<306>3695-3700

<307>2003-07-01

<308>AAO52683

<309>2005-04-14

<313>(31)..(124)

<400>10

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

Claims (5)

1, one group of HE4 gene synthetic primer is characterized in that: design and synthesize the PCR primer according to people HE4 gene order among the GenBank and in conjunction with the intestinal bacteria preference codon, PCR primer nucleotide sequence is formed:

P1：tggttgtgctactttttgttctttacctaatgataaagaaggttcttgtc

P2：ctaattgaggaaaattaatattaacttgaggacaagaaccttctttatca

P3：tctgaatgtgctgataatttaaaatgttgttctgctggttgtgctacttt

P4：ttgagaatcaacttgacattgatcacgacataaacctaattgaggaaaat

P5：ctgatcaaaattgtactcaagaatgtgtttctgattctgaatgtgctgat

P6：caaccattacgacaacatttcatttgaccaggacattgagaatcaacttg

P7：cgggatccgaaaaaactggtgtttgtcctgaattacaagctgatcaaaattgta

P8：ccgctcgagttattaaaaattaggagtaacacaagaaactttaccacaaccattacgacaa。

2, the HE4 gene of synthetic is characterized in that: according to the primer of claim 1 design, obtain the HE4 gene by overlapping PCR method, gene order is as follows:

1 gaaaaaactggtgtttgtcctgaattacaagctgatcaaaattgtactcaagaatgtgtt

61 tctgattctgaatgtgctgataatttaaaatgttgttctgctggttgtgctactttttgt

121?tctttacctaatgataaagaaggttcttgtcctcaagttaatattaattttcctcaatta

181?ggtttatgtcgtgatcaatgtcaagttgattctcaatgtcctggtcaaatgaaatgttgt

241?cgtaatggttgtggtaaagtttcttgtgttactcctaatttt?282。

3, HE4 protein expression is characterized in that: according to the described gene of claim 2, the synthetic HE4 gene that obtains is cloned in the pGEX-4T1 carrier by BamH I and Xho I restriction enzyme site, at expression in escherichia coli and be purified into reorganization GST-HE4 albumen.Obtain the pure product of HE4 albumen through zymoplasm cutting and purifying, its aminoacid sequence is formed:

E?K?T?G?V?C?P?E?L?Q?A?D?Q?N?C?T?Q?E?C?V?S?D?S?E?C?A?D?N?L?K?C

C?S?A?G?C?A?T?F?C?S?L?P?N?D?K?E?G?S?C?P?Q?V?N?I?N?F?P?Q?L?G?L?C

R?D?Q?C?Q?V?D?S?Q?C?P?G?Q?M?K?C?C?R?N?G?C?G?K?V?S?C?V?T?P?N?F。

4, HE4ELISA detection kit, its preparation method is: require 3 described protein HE4 immune animals to obtain polyclonal antibody and monoclonal antibody with containing right, with this polyclonal antibody as capture antibody, as detecting antibody, be assembled into the double-antibodies sandwich ELISA detection kit with monoclonal antibody.

5, the described test kit of claim 4, it is characterized in that: it can be used for the auxiliary diagnosis of human ovarian cancer.Particularly to the early stage auxiliary diagnosis of ovarian cancer patients.

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