CN101334410A - Method and reagent kit for detecting endometriosis - Google Patents

Abstract

The invention discloses a method for detecting endometriosis, which belongs to the medical laboratory field. The method for detecting the endometriosis judges whether a subject suffers from the endometriosis or not through the measurement of the expression level of GREM1 protein in endometrial tissue or serum. The method has the advantages that: the method firstly reveals the relationship between the expression of the GREM1 protein and the endometriosis, and further provides an effective marker for detecting the endometriosis. The method for detecting the endometriosis and a kit of the invention adopt the ELISA to carry out the quantitative measurement of the content of the GREM1 protein in the serum of the subject, the requirements on the pretreatment of samples is low, the pretreatment process of the samples is simple, the method can simultaneously detect a large number of samples, and the detection method is simple, convenient and practicable.

Description

A kind of method and kit that detects endometriosis

Technical field

The present invention relates to a kind of method and kit that detects endometriosis, belong to field of medical examination.

Background technology

(Edometriosis EM) is endometriosis beyond uterine cavity and causes pathology endometriosis, is a kind of common disease, frequently-occurring disease, causes pelvic pain, intercourse pain and infertile etc., has a strong impact on women's physical and mental health.The incidence of disease accounts for 30%-50% at 15%-20% in the infertile women.Its pathogenesis still imperfectly understands, and has many theories such as endometrial implantation theory, immunological theory, lymph and vein to send out theory, the living theory of coelomic epitheliumization etc., but all can not explain the morbidity of gynecopathy fully.

Fundamental research shows, plays an important role during vascularization and immunologic unusual pathology at EM take place.Sha Guihua etc. studies show that, compare with the capillary endothelium in non-EM source, and capillary endothelium membrane derived in EM patient is on the throne has shown the survival ability of obvious enhancing in the process that in-vitro separation is cultivated.Studies show that of molecular level compared with the inner membrance on the throne of non-EM normal control, and (GREM1, GenbankAF110137) expression in this cell obviously strengthens Gremlin 1.

GREM1 is bone morphogenetic protein (bone morphogenetic proteins, BMPs) one of antagonist family member, be one to contain the secretary protein of 184 amino acid residues, molecular weight is 20697Da, the conservative property that in the biological evolution process, has height, its amino acid sequence height homology between people and rat, mouse, chicken and Java genus.It can combine (especially BMP-2 ,-4 ,-7) with specific BMPs member, forms heterodimer, suppresses BMPs and its receptors bind, produces antagonism.It both had been present in endocytoplasmic reticulum and the golgiosome tube chamber, also was present in cell surface, linked to each other with cell membrane with the non-covalent bond form.Glycosylation and non-glycosylated two kinds of forms are arranged, and the two is the activity of energy antagonism BMPs all.

GREM1 mainly expresses in brephic histoorgan, can regulate the growth of limbs, suppresses that cartilage takes place and the apoptosis of cell, and the growth of histoorgans such as four limbs, nerve, kidney, lung, skin, feather, ear is played an important role.It also is present in the humans and animals tissue of manhood, and the expression of germlin is also arranged in the human multiple tissue, in small intestine, colon and embryo and brain high expressed is arranged, weak expression in Adult Human Brain, ovary, pancreas, prostate, skeletal muscle.In the research to adult's disease, think GREM1 and diabetic nephropathy, DRP, glaucoma, kidney fibrosis, pulmonary fibrosis, liver fibrosis, even the generation of tumour all there is substantial connection.GREM1 transforms with promoting epithelium-mesenchyma in these fibrotic diseases, and it is relevant to suppress epithelial cell regeneration.Research in the gynemetrics field mainly concentrates on, and it is expressed by egg mother cell and granular cell in ovary, by the effect modulability hormone generation of paracrine.Have or not the expression of GREM1 in the endometrial tissue, the irrelevant relevant report that at home and abroad there is no is arranged with endometriosis.

In view of above-mentioned new knowledge to gynecopathy morbidity, the present invention selects that to form relevant Protein G REM1 be research object with urging new vessels, has disclosed the relation of this albumen and gynecopathy first, and has successfully developed the method and the reagent of detection endometriosis.

Summary of the invention

The technical problem to be solved in the present invention is: a kind of method that detects endometriosis is provided.

Another technical matters that the present invention will solve is: a kind of enzyme-linked immunosorbent assay kit that is used to detect endometriosis is provided.

For achieving the above object, the present invention is by the following technical solutions:

A kind of method that detects endometriosis, the expression of GREM1 albumen judges whether to suffer from endometriosis in mensuration experimenter's endometrial tissue on the throne or the serum.The detection index of reference: during protein content in the peripheral blood serum 〉=700ng/ml, infer and suffer from endometriosis.

According to the common practise of this area, known that GREM1 albumen can be as the label of endometriosis, thereby can adopt several different methods to detect the diagnosis that its expression carries out disease.The method of described detection endometriosis can be Western Blot method, chemoluminescence method, euzymelinked immunosorbent assay (ELISA) or PCR method (preferred RT-PCR), first three methods is in the variation of the expression of protein level detection GREM1 albumen, and the PCR method can detect at gene level.

The present invention adopts protein immunoblotting method (Western blot analysis), has proved short angiogenesis factor--and GREM1 albumen has expression in the film in uterus.Further compared the difference of expression in endometriosis (EM) patient's endometrial tissue on the throne and non-endometriosis patient's the endometrial tissue, the result shows that the expression of GREM1 albumen in endometrium ectopia patient's endometrial tissue on the throne is higher than non-endometrium ectopia patient's endometrium.We are to the result of study of the GREM1 mRNA level of two groups of patients' endometrial tissue on the throne, be presented at that the GREM1 rna level obviously increases (P＜0.05) than non-EM patient in EM patient's the endometrial tissue on the throne, point out that the expression of GREM1RNA and protein level all exists in our EM patient and non-EM patient's the endometrial tissue on the throne, between two groups notable difference is arranged, exist more GREM1 in EM patient's the endometrial tissue on the throne.Carry out Western blotting after wherein two women's serum is removed high-abundance proteins, wherein EM patient obviously raises than non-EM patient's band gray-scale value.This result adopts euzymelinked immunosorbent assay (ELISA) to detect disease for us and the exploitation enzyme-linked immunologic detecting kit provides foundation.

Preferred enzyme linked immunosorbent assay of the present invention is measured the expression of GREM1 albumen in experimenter's serum, and step is as follows:

1. bag quilt: with the phosphate buffer of coating buffer 0.02mol/L pH 7.2 the GREM1 monoclonal antibody being diluted to protein content is 1～10 μ g/ml, adds 100ul in each reacting hole of polystyrene board, puts 4 ℃ and spends the night; Discard solution in the hole next day,, pat dry with cleansing solution PBST washing 1～3 time;

2. sealing: add confining liquid (5% skimmed milk) 200ul, 37 ℃ were sealed 2 hours, with lavation buffer solution PBST washing 1～3 time, discarded solution in the hole, patted dry;

3. application of sample: every hole adds 50ul sample to be checked (undiluted serum) and the final concentration that dilutes with 0.5% skimmed milk is the GREM1 polyclonal antibody 50ul of 0.2ug/ml, hatch 30min jointly for 37 ℃, with lavation buffer solution PBST washing 3～5 times, discard solution in the hole, pat dry; Typical curve: every hole adds the 50ul standard items, concentration is 0ppb, 100ppb, 200ppb, 400ppb, 800ppb and 1200ppb, with the final concentration with 0.5% skimmed milk dilution be the GREM1 polyclonal antibody 50ul of 0.2ug/ml, hatch 30min jointly for 37 ℃, with lavation buffer solution PBST washing 3～5 times, discard solution in the hole, pat dry;

4. add enzyme labelled antibody: every hole adds the HRP-goat anti-rabbit igg 100ul with the dilution in 1: 3000 of 0.5% skimmed milk, 37 ℃ hatch 30min after PBST wash 5 times, discard solution in the hole, pat dry;

5. chromogenic reaction: every hole adds TMB (tetramethyl benzidine) and uses liquid 100ul, colour developing 15min;

6. cessation reaction: every hole adds 50ul 2mol/L H ₂SO ₄Cessation reaction;

7. the result judges: on the ELISA detector, in the 450nm place, measure the OD value of each reacting hole with zeroing back, blank hole; Standard items absorbance with variable concentrations is that horizontal ordinate, GREM1 concentration are that ordinate carries out linear fit, gets equation of linear regression, according to protein content in the regression equation working sample.During serum albumin content 〉=700ng/ml, infer and suffer from endometriosis.

The antibody that the present invention uses can obtain by commercial sources, also can prepared in laboratory.Polyclonal antibody and MONOCLONAL ANTIBODIES SPECIFIC FOR method are techniques well known.

A kind of enzyme linked immunological kit that detects endometriosis is made up of following reagent, and 2～8 ℃ keep in Dark Place:

1.48 or 96 hole polystyrene plates;

2.GREM1 mouse monoclonal antibody (H00026585-M07), Abnova company;

3.GREM1 rabbit polyclonal antibody (sc-28873), santa cruz company;

4. enzyme labelled antibody: horseradish peroxidase-labeled goat anti-rabbit igg, santa cruz company;

5.GREM1 standard items: with the PBS dilution, concentration is 0ppb, 100ppb, 200ppb, 400ppb, 800ppb and 1200ppb; (recombinant mouse GREM1,956-GR/CF), R﹠amp; D company;

6. coating buffer: pH 7.2, concentration are the phosphate buffer PBS of 0.02mol/L

7.10 doubly concentrated cleaning solution PBST:pH 7.4, concentration 1.5mol/L are with KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, NaCl 8.0g, KCl 0.2g and TWeen-200.5ml are dissolved in 100ml distilled water; Use preceding water or deionized water to be diluted to final concentration for 10 times and be 0.15mol/L;

8. confining liquid: 5% skimmed milk is dissolved in 100ml cleansing solution PBST with the 5g skimmed milk power;

9. antibody diluent: 0.5% skimmed milk is dissolved in 100ml cleansing solution PBST with the 0.5g skimmed milk power;

10. substrate colour developing liquid: tetramethyl benzidine (10mg/5ml absolute ethyl alcohol) 0.5ml, 0.75%H ₂O ₂32 μ l, pH5.5 substrate buffer solution 10ml.Wherein the composition of substrate buffer solution is: 0.1M citric acid (19.2g/L) 24.3ml, 0.2M Na ₂HPO ₄12H ₂O (71.7g/L) 25.7ml and 50ml distilled water;

11. stop buffer: 2M H ₂SO ₄, distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml.

Advantage of the present invention: the present invention has disclosed the relation of GREM1 expressing quantity and endometriosis first, thereby a kind of label of effective detection endometriosis is provided.The present invention detects the method and the kit of endometriosis, adopt the content of GREM1 albumen in the ELISA quantitative measurement experimenter serum, low to the pre-treatment requirement of sample, sample pretreatment process is simple, energy is the fast detecting gross sample simultaneously, and detection method is simple and easy to do.

The invention will be further described below in conjunction with the drawings and specific embodiments; it is not limitation of the invention; according to prior art well known in the art; embodiments of the present invention are not limited to this; therefore all this areas of having done according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.

Description of drawings

Fig. 1 is for detecting the result of GREM1 in the endometrial tissue on the throne (EM group and non-EM group) with western blot.

Fig. 2 is the contrast of GREM1 albumen relative expression quantity in EM and non-EM patient endometrium on the throne.

Fig. 3 is the GREM1 result in the western blot detection serum.

Fig. 4 is the contrast of 1 routine EM patient and 1 routine non-EM patients serum GREM1 relative expression quantity.

Fig. 5 detects the GREM1 protein standard substance for Western Blot.

Fig. 6 is for measuring the typical curve of GREM1 albumen in the serum with double antibodies sandwich indirect enzyme-linked immunosorbent method.

Fig. 7 is the concentration of GREM1 in EM group and the non-EM group serum.

Fig. 8 is the comparison of gremlin content in non-EM group propagation phase and the EM group propagation phase serum.

Embodiment

The correlativity of embodiment 1:GREM1 albumen and endometriosis

One. material

(1) experimental subjects

1. tissue specimen: 16 examples of drawing materials altogether all are that it is endometriosis patient 10 examples, non-endometriosis patient 6 examples that operation confirms the undergo surgery patient of treatment of my institute (BJ Union Hospital).EM organizes and non-EM group patient's the standard of including in is, child-bearing period menstruation rule, and cycle 25-34 days, there are not other endocrine, immunity and metabolic disease, do not accept hormone therapy in the operation first trimester.Get endometriosis patient's endometrial tissue on the throne and non-endometriosis patient's endometrial tissue, put into liquid nitrogen and preserve.

2. blood preparation: get 1 example endometriosis disease patient A (26 years old, the skilful capsule of bilateral), 1 routine non-endometriosis patient's (43 years old, follicular cyst of ovary and simple cyst) serum, both all confirm at the court gynemetrics underwent operative pathology.Extracting vein blood 2ml before the art, the EDTA anti-freezing, 3000rpm, 10 minutes, get serum and be sub-packed in the Eppendorf centrifuge tube, be stored in-70 ℃ of refrigerators.

(2) experiment material

1. main agents:

One is anti-: GREM1 mouse monoclonal antibody, Abnova company (H00026585-M07), GREM1 rabbit polyclonal antibody, santa cruz company (sc-28873), mouse β-actin IgG, Shanghai brilliant U.S. Bioisystech Co., Ltd

Two is anti-: horseradish peroxidase-labeled goat anti-rabbit igg, santa cruz company

The horseradish peroxidase-labeled goat anti-mouse IgG, China fir Bioisystech Co., Ltd in Beijing

The standard items of GREM1, R﹠amp; (recombinant mouse GREM1,956-GR/CF) total protein extracts kit, Beijing Puli's lema gene technology company limited in D company

Remove albumin and IgG kit, Amersham Biosciences company

BCA protein assay kit, Pierce company

2. other reagent:

Tris Base，promega

Glycocoll, Beijing chemical reagents corporation

SDS，sigma

AP，sigma

Skimmed milk power, santa cruz

NaCl Beijing chemical reagents corporation

Methyl alcohol, Beijing Century Red Star chemical industry Ltd

Absolute ethyl alcohol, Beijing chemical reagents corporation

TEMED，invitrogen

Tween 20，ameresco

Bovine serum albumin(BSA) (BSA), Beijing Fang Run biotech firm

A, B tracing liquid, Santa cruz company

Contrasting power, Beijing Puli's lema gene technology company limited

Fixing powder, Beijing Puli's lema gene technology company limited

3. equipment and instrument:

96 orifice plate Beijing Puli's lema gene technology company limiteds

37 ℃ of incubators

Microplate reader, Eppendoff, Germany

Magnetic stirring apparatus Shanghai Si Le instrument plant

Accurate liquid-transfering gun Eppendorf, Germany

Half-dried commentaries on classics film instrument, Amersham pharmacia biotek

High speed freezing centrifuge, Eppendorf, Germany

Water bath, the U.S. scientific instrument of Beijing Orient crystalline substance company limited

Table model high speed centrifuge, Anting Scientific Instrument Factory, Shanghai

TS-1 type decolorization swinging table, its woods Bel instrument Manufacturing Co., Ltd of Haimen City, Jiangsu

Electronic balance, Shanghai plum Teller-Tuo benefit Instr Ltd.

Two permanent electrophoresis apparatuses, Beijing Orient instrument plant

Two vertical electrophoresis grooves, Beijing Liuyi Instrument Factory

The X ray camera obscura, Shoutou Yuehua Medical Apparatus Factory Co., Ltd.

The gel imaging analysis system, syngene

Two. experimental technique

Protein immunoblotting method (Western blot analysis) experimental technique is with reference to the method for " molecular cloning experiment guide " (third edition) related Sections and Molecular Biology Lab of preclinical medicine institute of China Concord Medical Science University.

(1) liquid dosage

1.10%SDS:SDS 10g, adding distil water are to 100ml, and room temperature preservation is dissolved in 50 ℃ of water-baths down.

2.10% Ammonium Persulfate 98.5 (AP): AP 0.1g adds ultrapure water 1.0ml, after the dissolving, and-20 ℃ of preservations.

3.1.5mol/L TrisHCl (pH8.8): Tris (MW121.14) 45.43g adds ultrapure water 200ml, after the dissolving, transfers pH to 8.8 with concentrated hydrochloric acid, is settled to 250ml with ultrapure water at last, preserves under the room temperature.

4.0.5mol/L TrisHCl (pH6.8): Tris (MW121.14) 15.14g adds ultrapure water 200ml, after the dissolving, transfers pH to 6.8 with concentrated hydrochloric acid, is settled to 250ml with ultrapure water at last, preserves under the room temperature.

5.100mg/ml bovine serum albumin(BSA) (BSA): BSA 0.1g, add ultrapure water 1ml, when making the protein standard curve, carry out 100 times with ultrapure water and be diluted to 1mg/ml ,-20 ℃ of preservations.

6.30% acrylamide/methylene acrylamide: acrylamide 29g, methylene acrylamide 1g adds water 60ml dissolving back moisturizing to 100ml, filter paper filtering, 4 ℃ of storages of lucifuge.

7. (5X) SDS sample-loading buffer: 0.5mol/L TrisHCl (pH6.8) 2.5ml, two sulphur uncle sugar alcohols (DTT, MW154.5) 0.39g, SDS 0.5g, bromophenol blue 0.025g, glycerine 2.5ml behind the mixing, is sub-packed in the 1.5ml centrifuge tube 4 ℃ of preservations.

8. electrophoresis liquid damping fluid (1X): Tris (MW121.14) 3.03g, glycocoll (MW75.07) 18.77g, SDS 1g, adding distil water is to 1000ml.

9. transfering buffering liquid (1X): glycocoll (MW75.07) 2.9g, Tris (MW121.14) 5.8g, SDS0.37g, methyl alcohol 100ml, adding distil water is to 1000ml.

10.TBST damping fluid: 1mol/L TrisHCl (pH8.0) 100ml, NaCl 9g, Tween 201ml, adding distil water is to 1000ml.

11. confining liquid: skimmed milk power 5g, add TBST 100ml, 4 ℃ of preservations were no more than for 1 week.

12. developer solution (1X): will dissolve 4 ℃ of preservations of lucifuge sealing among developer solution powder (Beijing Puli's Lay biotech company) adding tap water (the being heated to 50 ℃) 500ml.

13. stop bath (1X): will dissolve lucifuge sealing room temperature preservation among stop bath powder (Beijing Puli's Lay biotech company) adding tap water (the being heated to 50 ℃) 500ml.

(2) experimental procedure:

1. protein sample preparation:

(1) tissue sample preparation: the total protein that adopts Puli's Lay company to provide extracts kit.

Get the about 100mg of fritter endometrium (totally 16 parts) respectively, shred the back and add the 1ml lysate, put into glass homogenizer, manual homogenate on ice 15 times; Get the 0.5ml tissue homogenate and be transferred in the 1.5ml centrifuge tube, add the abundant mixing of 1ml extraction agent, 4 ℃ leave standstill 10min, rock once in a while; Behind 4 ℃, the centrifugal 10mins of 10000rpm, solution is divided into two-phase up and down, is protein film in the middle of the two-phase.The liquid of sucking-off levels obtains protein film; After opening wide dry 10min, add the 1%SDS soluble protein film of 200ul, centrifugal 3000rpm 3min removes not dissolved matter.The protein sample of gained is in-70 ℃ of preservations.

(2) blood serum sample preparation: that adopts that Amersham Biosciences company produces removes albumin and IgG kit, and this method can be removed in the serum＞95% albumin and＞90% IgG.

In 2 1.5ml centrifuge tubes, add each 15ul of serum of EM patient's 1 example and non-EM patient's 1 example respectively, add 750ul resin suspension respectively, room temperature is fully mixed to few 30min, the about 250bpm of concussion speed; Cut off the lower end of Filter column, and put into micro-centrifuge tube, the potpourri of abundant mixing is all moved in the Filter column cover lid; Behind 4 ℃, the centrifugal 5min of 10000rpm, collect filtrate, every part of serum obtains 300ul filtrate approximately ,-70 ℃ of preservations.

2. the mensuration of protein content (BCA method)

(1) gets 0.1g bovine serum albumin(BSA) (BSA) and be dissolved in the 1ml distilled water, obtain the 100mg/ml bovine serum albumin(BSA), be diluted to 1mg/ml BSA its 100 times.-20 ℃ of preservations.

(2) get 7 1.5ml centrifuge tubes, be labeled as respectively: 0mg/ml, 0.025mg/ml, 0.05mg/ml, 0.1mg/ml, 0.2mg/ml, 0.4mg/ml, 0.8mg/ml.

(3) preparation standard product: in each pipe, add all ingredients by table 1.

Table 1: the typical curve of working sample protein concentration

Concentration mg/ml	0	0.025	0.05	0.1	0.2	0.4	0.8
								1mg/ml BSA	0μl	25μl	50μl	100μl	200μl	400μl	800ul
ddH20	1ml	975μl	950μl	900μl	800μl	600μl	200ul

(4) sample is surveyed concentration (having extracted the histone liquid dilution 20-40 behind the total protein doubly, 20 times of the serum samples diluted after having removed albumin and IgG) with proper proportion dilution back.

(5) on 96 orifice plates, every duplicate samples and standard items are got 25ul and are added 200ulBCA working fluid (working fluid composition--A liquid: B liquid (V/V)=50: 1).

(6) behind the mixing, hatch 30min for 37 ℃.After being cooled to room temperature, 560nm surveys the OD value on microplate reader, makes the absorbance value typical curve of protein concentration, and determines the protein concentration of testing sample in the BSA typical curve.

3.SDS-PAGE electrophoresis

Alignment tightens after putting into the insulation adhesive tape in the middle of (1) two glass plate, and vertical card is prepared encapsulating on the top of the shelf.Earlier have or not leakage glue, alcohol as having the glue of leakage, toppling over, and blot with thieving paper with alcohol testing.

(2) join 12% separation gel by table 2, add TEMED at last, shake up encapsulating immediately behind the adding TEMED.It is fast that encapsulating speed is wanted, and avoids bubble.On glue, gently add one deck 75% alcohol immediately after adding separation gel, make the glue face smooth.

(3) glue to be separated fully solidifies back (room temperature is solidified about 30min), discards the alcohol on glue upper strata and with thieving paper alcohol is blotted.

(4) join 5% concentrated glue by table 2, shake up the back encapsulating immediately after adding TEMED.With remaining space fill concentrate glue after, the Teflon comb inserted concentrate in the glue.Room temperature is solidified 15min.

(5) by the time after concentrating gelling admittedly, the insulation adhesive tape of dismantling pours into electrophoresis liquid, and note draining the bubble of glue bottom, electrophoresis liquid there was not the gel top layer.Extract the Teflon comb.

(6) behind the finished white content of survey, the protein sample of sample in the preparation.The sample total protein concentration of sample is 100 μ g on each hole, add 5 * SDS sample-loading buffer to final concentration and be 1 * and, last sample volume is 25ul.Guarantee each swimming lane total protein concentration and go up the sample volume all consistent.Before the last sample with sample and dye albumen marker in advance and in boiling water, boil 5min and make albuminous degeneration.

(7) go up sample: every batch of histone sample runs three glue simultaneously, and three films that obtain are hatched with GREM1 polyclonal antibody, GREM1 monoclonal antibody and confidential reference items β-actin respectively.

(8) electrophoresis: concentrate glue constant voltage 130V, about 10min, separation gel constant voltage 160V, about 110min.Electrophoresis has just been run out of and can have been stopped to bromjophenol blue.

Table 2:SDS-PAGE electrophoretic separation glue concentrates the glue prescription

4. semidry method is changeed film

(1) changes a film and need prepare 6 filter paper and 1 pvdf membrane.Pvdf membrane soaks in methyl alcohol behind the 15min with filter paper, glue balance 10min in electrotransfer liquid.Film＞glue is avoided causing short circuit after the filter paper contact on both sides.

(2) stripping glue: in the enamel tray that fills electrotransfer liquid, glass plate is prized gently, will concentrate glue and scrape off gently, and size is cut glue as required.

(3) on half-dried electroporation, make " sandwich ".If bubble, roll wherein bubble in the process with glass rod.Do not move again after the stationkeeping of film and glue.

(cathode plane)

Three wetting Whatman 3M filter paper

Wetting SDS-PAGE gel

Wetting PVDF transfer film

Three wetting Whatman 3M filter paper

(anode surface)

(4) cover the lid of half-dried electroporation, change two films simultaneously and when above, need to place weight on the lid.Electricity commentaries on classics condition: constant current, the total area (cm^2) * 0.8 of electric current (mA)=film; 80mim.

(5) electricity changes near the back that the finishes albumen marker that dyes in advance of visible desired molecule amount has changeed on the film, and shear angle is with the mark front on film.

5. immune response

(1) film that takes a turn for the better is soaked with TBST after, move in the polybag of the sealing that contains confining liquid, shake on the decolorization swinging table under the room temperature sealing 2h.

(2) will seal good film and put into the polybag of sealing, one anti-ly is diluted to debita spissitudo (dilution in 1: 500 of GREM1 mouse monoclonal antibody, the dilution in 1: 800 of GREM1 rabbit polyclonal antibody, mouse polyclone β-actin antibody dilution in 1: 1000) with confining liquid; Drain bubble; Hatch 3h under 4 ℃ of night incubation or the room temperature on the Universal table, at room temperature wash 10min*3 time on the decolorization swinging table with TBST.

(3) the same method is hatched two anti-ly, and two anti-ly are diluted to debita spissitudo (goat anti-mouse igg dilution in 1: 1000, goat anti-rabbit igg dilution in 1: 2000) with confining liquid, hatch 1.5h under the room temperature after, at room temperature wash 10min*3 time on the decolorization swinging table with TBST.

6. chemiluminescence, development, photographic fixing

(1) with film slightly control lie against on the parafilm after doing, chemiluminescence agent A and two kinds of reagent equal-volumes of B of Santa Cruz are mixed the back point on film; Behind the 1min, slightly control is dried with film, moves in the polybag and wraps, and puts into X-mating plate folder and fixed position.

(2) in the darkroom, red light source takes out the X-ray sheet down, cuts out suitable size; Open X-ray sheet folder, the X-ray sheet is placed on the film,, can not move in case put.

(3) shut X-ray sheet folder, pick up counting, the film time shutter of having hatched the GREM1 polyclonal antibody is 1min, and the film of having hatched the GREM1 monoclonal antibody is 2min with the film time shutter of having hatched β-actin;

(4) after exposure is finished, take out the X-ray sheet, immerse in the developer solution rapidly and develop, the about 2min of development time, wait to occur obvious band after, stop development at once.In clear water behind the flush away developer solution, the X-ray sheet is immersed in the stop bath at once, fixing time with film transparent till; After washing away residual stop bath with tap water, dry film under the room temperature.

7. gel images analysis

Film is scanned or take pictures, with gel images disposal system evaluating objects band.Resist the band that obtains for the GREM1 monoclonal antibody to do to compare with one than the gray-scale value that obtains with the band of β-actin.

Three. the result:

1. referring to Fig. 1, EM patient's endometrial tissue on the throne 10 examples, non-EM patient's endometrial tissue 6 examples, each 1 example of EM patient and non-EM patient's serum, adopt the protein immunoblot result of GREM1 monoclonal antibody to obtain a band, molecular weight is 47KD; Adopt the protein immunoblot result of GREM1 polyclonal antibody to obtain two bands, molecular weight is respectively 47KD and 60KD.

2. referring to Fig. 2, gel images is analyzed the band gray-scale value, the GREM1 band at the 47KD place of monoclonal antibody gained and the band gray-scale value of β-actin are made ratio: EM group GREM1 band is 1.92 ± 0.41 with the ratio of the band gray-scale value of β-actin, and it is 0.89 ± 0.26 that non-EM organizes.There were significant differences for two groups of data, P＜0.05.

3. referring to Fig. 3, EM patient's serum 1 example compares with GREM1 protein expression shown in non-EM patient's the serum 1 routine Western Blot, adopts the protein immunoblot result of GREM1 monoclonal antibody to obtain a band, and molecular weight is 47KD; Adopt the protein immunoblot result of GREM1 polyclonal antibody to obtain two bands, molecular weight is respectively 47KD and 60KD.

4. referring to Fig. 4, there were significant differences in the contrast of EM patient's 1 example and non-EM patient's 1 routine serum GREM1 relative expression quantity.

5. referring to Fig. 5, REM1 protein standard substance Western Blot result: resist as one with monoclonal antibody, polyclonal antibody respectively and hatch, all obtain the band that molecular weight is the 20KD size, consistent with GREM1 standard items molecular weight size, prove two kinds of antibody all can with GREM1 protein standard substance generation antigen-antibody reaction.

Four. discuss:

Adopt the protein immunoblot result of GREM1 monoclonal antibody can both and only can obtain a band, molecular weight is 47KD; Adopt the protein immunoblot result of GREM1 polyclonal antibody all can obtain two bands, molecular weight is respectively 47KD and 60KD.The molecular weight that adopts two kinds of antibody to obtain is that the band of 47KD size is common.This experiment all detects the band that molecular weight is 47KD with GREM1 monoclonal antibody and polyclonal antibody, and with the experiment that sample on the GREM1 standard items carries out western blot also confirmed these two kinds of antibody all can with GREM1 protein standard substance generation antigen-antibody reaction.Therefore, have reason to think that molecular weight is that the band of 47KD is a GREM1 albumen.

Five. conclusion:

It is short that angiogenesis factor--GREM1 albumen has expression in the film in uterus.Its expression in endometrium ectopia patient's endometrial tissue on the throne is higher than non-endometrium ectopia patient's endometrium.

Embodiment 2: set up the double antibodies sandwich indirect elisa method and detect GREM1 level among the EM patients serum

One. material

(1) experimental subjects:

Blood preparation: (Concord Hospital) gynemetrics underwent operative turns out to be endometriosis patient and non-endometriosis patient totally 37 examples in the court to choose year April in January, 2007 to 2007, and wherein 23 examples are organized for EM, age 17-49 year.Non-EM organizes 14 examples, comprises teratoma, ovary simple cyst, follicular cyst of ovary, seromucus Combination cystadenoma, age 20-48 year.EM organizes 34 ± 8.449 years old age, and non-EM organizes 31.78 ± 6.782 years old age, and two groups of ages are compared no statistical significant difference, P＞0.05.Extracting vein blood 2ml before the art, the EDTA anti-freezing, 3000rpm, 10 minutes, get serum and be sub-packed in the Eppendorf centrifuge tube, be stored in-70 ℃ of refrigerators.

(2), experiment material

1. main agents:

GREM1 mouse monoclonal antibody (H00026585-M07), Abnova company

GREM1 rabbit polyclonal antibody (sc-28873), santa cruz company

The horseradish peroxidase-labeled goat anti-rabbit igg, santa cruz company

The GREM1 standard items (recombinant mouse GREM1,956-GR/CF), R﹠amp; D company

2. other reagent:

Na ₂CO ₃, NaHCO ₃, KH ₂PO ₄, Na ₂HPO ₄12H ₂O, NaCl, KCl, Beijing chemical reagents corporation

TWeen-20，ameresco

Skimmed milk power, santa cruz

BSA, Beijing Fang Run biotech firm

Citric acid, Beijing chemical reagents corporation

TMB (tetramethyl benzidine), Beijing chemical reagents corporation

The concentrated sulphuric acid (98%)

(3) equipment and instrument:

Enzyme connection detector, BioTek company,

Automatic washer, BioTek company.

96 hole polystyrene microdetermination plates, the bright magnificent company limited of Shenzhen gold.

37 ℃ of incubators

Accurate liquid-transfering gun, Eppendorf, Germany

Table model high speed centrifuge, Anting Scientific Instrument Factory, Shanghai

Electronic balance, Shanghai plum Teller-Tuo benefit Instr Ltd.

Two. experimental technique

(1) liquid dosage

1. (pH 9.6,0.05M): Na for carbonate buffer solution CB ₂CO ₃1.59g, NaHCO ₃2.93g, adding distil water 1000ml (4 ℃ of preservations).

2. (pH 7.2,0.02mol/L): KH for phosphate buffer PBS ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, adding distil water 1000ml (4 ℃ of preservations).

3. (pH 7.4,0.15M): KH for lavation buffer solution PBST ₂PO ₄0.2g, Na ₂HPO ₄12H2O 2.9g, NaCl8.0g, KCl 0.2g, TWeen-20 0.5ml, adding distil water 1000ml (4 ℃ of preservations).

4. confining liquid: skimmed milk power 5 grams add PBST 100ml.

5.1%BSA: bovine serum albumin(BSA) (BSA) 1 gram adds PBST 100ml.

6. antibody diluent: skimmed milk power 0.5 gram adds PBST 100ml.

7. substrate buffer solution (pH 5.5): 0.1M citric acid (19.2g/L) 24.3ml, 0.2M Na ₂HPO ₄12H ₂O (71.7g/L) 25.7ml, adding distil water 50ml (4 ℃ of preservations).

8.TMB (tetramethyl benzidine) uses liquid: TMB (10mg/5ml absolute ethyl alcohol) 0.5ml, substrate buffer solution (PH5.5) 10ml, 0.75%H ₂O ₂32 μ l.

9. stop buffer (2M H2SO4): distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml.

(2) experimental procedure

1. the foundation of double-antibody sandwich indirect ELISA method:

(1) wraps by 96 hole polystyrene microdetermination plates with GREM1 monoclonal antibody 100ul, put 4 ℃ and spend the night.Dry liquid PBST washing 1 time, pat dry.

(2) add confining liquid 200ul, 37 ℃ were sealed 2 hours, and PBST washing 1 time throws away liquid and pats dry.

(3) add antigen 50ul to be checked and GREM1 polyclonal antibody 50ul, hatch 30min jointly for 37 ℃, PBST washing 5 times throws away liquid and pats dry.

(4) add HRP-goat anti-rabbit igg 100ul again, hatch behind the 30min PBST washing 5 times, throw away liquid and pat dry for 37 ℃.

(5) add substrate TMB 100ul colour developing 15min, use 2mol/L H ₂SO ₄50ul stops, and is determined at the OD value of 450nm.

2. the condition optimizing of detection method:

(1) monoclonal antibody IgG wraps by concentration:

Monoclonal antibody is done dilution in 1: 100,1: 500,1: 1000,1: 3000,1: 5000,1: 10000 and is wrapped by polystyrene board, every hole 100ul, fixed standard product concentration 300ng/ml, polyclonal antibody were by dilution in 1: 1000, and the HRP-goat anti-rabbit igg uses by dilution in 1: 3000.

(2) bag is cushioned the selection of liquid:

Monoclonal antibody is done dilution in 1: 1000, use respectively carbonate buffer solution (pH9.6,0.05mol/L), (pH 7.2 for phosphate buffer, 0.02mol/L) and distilled water be cushioned liquid as bag, each wraps by under the condition, does a blank well and the standard items hole that concentration is 100ng/ml.Polyclonal antibody was by dilution in 1: 1000, and the HRP-goat anti-rabbit igg uses by dilution in 1: 3000.

(3) polyclonal antibody IgG reaction density:

(pH 7.2 for monoclonal anti body and function phosphate buffer, 0.02mol/L) do to wrap quilt after the dilution in 1: 1000, fixed standard product concentration 300ng/ml, polyclonal antibody reacts by dilution in 1: 500,1: 1000,1: 3000,1: 5000,1: 10000, and the HRP-goat anti-rabbit igg uses by dilution in 1: 3000.

(4) determining of optimal reaction time:

Under equal conditions carry out double antibodies sandwich indirect ELISA experiment, get each step reaction time respectively and be 30,45,60min, measure the OD value in the standard items hole of 300ng/ml.

(5) selection of confining liquid and antibody diluent:

Confining liquid is selected three kinds of 5% skim milks, 1%BSA, 5% skim milk+0.5% gelatin respectively, and be 2 hours off-period.Antibody diluent selects skimmed milk concn to be respectively 5%, 3%, 1%, 0.5% respectively.Do a blank well and the standard items hole that concentration is 100ng/ml under every kind of condition.

(6) determining of serum diluting multiple:

The serum of getting 2 EM patients and 2 non-EM women all by * 1, * 20, * 100 dilute.Monoclonal antibody is done dilution in 1: 1000, and (pH 7.2,0.02mol/L) make bag and are cushioned liquid, and polyclonal antibody was by dilution in 1: 1000, and the HRP-goat anti-rabbit igg uses by dilution in 1: 3000 for phosphate buffer.

Three, experimental result

(1) optimum reaction condition of double-antibody sandwich indirect ELISA method is determined:

1. monoclonal antibody IgG the best is wrapped determining by concentration:

As shown in table 3, different monoclonal antibody bags by the concentration situation under, fix other reaction conditionss, survey the OD value of standard items concentration 300ng/ml.As seen (1ug/ml) OD value is the highest when 1: 1000 times of dilution of monoclonal antibody, wraps best by effect.

Table 3: different monoclonal antibody bags is by the OD value under the concentration situation

The monoclonal antibody bag is by concentration (ug/ml)	0.1 ug/ml	0.2 ug/ml	0.33 ug/ml	1 ug/ml	2 ug/ml	10 ug/ml
							Standard items OD value (300ng/ml)	0.212	0.503	0.559	0.776	0.682	0.548
Blank OD value	0.098	0.120	0.076	0.091	0.410	0.172

2. bag is cushioned determining of liquid:

As shown in table 4, by under the environment, other reaction conditions unanimities are surveyed the OD value in blank well and standard items hole at different bags.As seen use pH 7.2, during the phosphate buffer of 0.02mol/L, blank well OD value is minimum, with the standard items hole of 100ng/ml notable difference is arranged.

Table 4: the OD value when selecting different bags to be cushioned liquid

	Carbonate buffer solution pH 9.6	Phosphate buffer pH 7.2	ddH 2O
				Blank OD value	0.305	0.072	0.197
Standard items OD value (100ng/ml)	0.198	0.264	0.301

3. polyclonal antibody IgG optimum response concentration is definite:

As shown in table 5, standard items concentration is 300ng/ml, adds the polyclonal antibody of variable concentrations, and when fixing other reaction conditionss, as seen (0.2ug/ml) OD value is the highest when 1: 1000 times of dilution of polyclonal antibody, and it is best to detect effect.

Table 5: add the OD value under the different how anti-concentration situations

Many anti-concentration ug/ml	0.02	0.04	0.07	0.2	0.4
						Standard items 300ng/ml OD value	0.235	0.336	0.594	0.769	0.522

4. the optimal reaction time is definite:

Get each step reaction time and be 30,45,60min, the OD value of measuring the standard items hole of 300ng/ml is respectively 0.765,0.771,0.693, difference is not remarkable, so selects the reaction time of 30min as each step.

5. confining liquid and antibody diluent is definite:

By table 6 as seen, be 5% skimmed milk at confining liquid, when being 0.5% skimmed milk, antibody diluent can guarantee that the blank well background is low and with the standard items hole of 100ng/ml maximum difference is arranged.

Table 6: the selection of confining liquid and antibody diluent

6. serum diluting multiple is definite:

According to 4 parts of serum results as seen: when serum did not dilute, therefore EM patient and non-EM patient's the different maximum of OD value difference selected * 1 as the suitableeest serum dilution.

Table 7: the selection of serum diluting multiple

	×1	×20	×100
				EM patient A	2.304	1.967	0.933
The EM patient B	1.647	1.142	0.866
				Non-EM patient a	1.188	0.961	0.758
Non-EM patient b	1.536	1.334.	0.871

7. according to above-mentioned condition optimizing step, determined that the optimum condition of GREM1 in the double antibodies sandwich indirect ELISA method detection serum is:

(1) use the GREM1 monoclonal antibody as capture antibody, bag is by elisa plate (concentration is 1ug/ml), and 4 ℃ are spent the night, and it is that (pH 7.2,0.02mol/L) for phosphate buffer that bag is cushioned liquid;

(2) every hole adds the sealing of 5% skimmed milk;

(3) every hole GREM1 polyclonal antibody (concentration is 0.2ug/ml) of adding 50ul sample to be checked (serum does not dilute) and 50ul is hatched jointly;

(4) GREM1 polyclonal antibody and HRP-goat anti-rabbit igg (dilution in 1: 3000) all dilute with 0.5% skimmed milk;

(5) except sealing and adding the substrate colour developing, all the other respectively go on foot the reaction time is 30min.

(2) testing result of GREM1 among the patients serum:

1. production standard curve:

Table 8: typical curve

Standard items concentration	0ppb	100ppb	200ppb	400ppb	800ppb	1200ppb
							The OD value	0.045	0.411	0.782	1.330	1.984	2.513

With the absorbance is that horizontal ordinate, GREM1 concentration are that ordinate carries out linear fit, and equation of linear regression is y=416.49x, and linearly dependent coefficient is 0.9357.Typical curve is consulted Fig. 6.

2. the level of GREM1 in the detection serum, the horizontal indifference of GREM1 in EM group and the non-EM group serum

Detect the serum of 22 routine EM patients and 14 routine non-EM patients (follicular cyst of ovary, ovary simple cyst, oviduct mesentery tumour, ovary maturity cystic teratoma).Adopt SSPS software package (version 12.0) to do data statistic analysis.EM group serum GREM1 protein content is 685.4 ± 249.5ng/ml, and non-EM group is 557.2 ± 140.5ng/ml, EM group patients serum's GREM1 content and non-EM group serum GREM1 content there was no significant difference (P=0.203).The result consults Fig. 7.

The testing result of GREM1 relatively in table 9:EM group and the non-EM group serum

Group	The example number	Serum GREM1 protein content ± SD
			The EM group	22/36	685.4±249.5
The non-EM group	14/36	557.2±140.5

EM group of participating in the experiment and non-EM group total number of persons are 37 people, and wherein an example is a stomach wall type endometriosis, not within statistics.

3. the GREM1 level has significant difference in non-EM group propagation phase and the EM group propagation phase serum

Patient's phase during endometrial cycle is divided into propagation phase and secretory phase again respectively with EM group and non-EM group, and carries out comparison between any two when pressing blood, and non-EM group propagation phase and EM organize propagation has significant difference between the phase, P＜0.05.There was no significant difference between propagation phase and secretory phase in the non-EM group, P=0.284.There was no significant difference between propagation phase and secretory phase in the EM group, P=0.566.Non-EM group secretory phase and EM there was no significant difference between the group secretory phase, P=0.537.The result consults Fig. 8.

The GREM1 level has significant difference in table 10:non-EM group propagation phase and the EM group propagation phase serum

	The example number	Serum GREM1 protein content x ± SD
			Non-EM organizes the propagation phase *	6/30	516.9±95.7
Non-EM organizes the secretory phase	6/30	603.4±158.0
			EM organizes the propagation phase *	13/30	678.1±224.3
EM organizes the secretory phase	5/30	741.1±298.3

The EM that participates in the experiment group and non-EM group total number of persons are 37 people, and it is indefinite wherein to classify mutually during inner membrance, and non-EM organizes 2 examples, and EM organizes 4 examples, not within this adds up.

Four. discuss: the level of finding non-EM group propagation phase and EM group propagation serum GREM1 between the phase in the experiment has significant difference, P＜0.05.This result is understood that the propagation phase of film, except inner membrance on the throne, to also have the inner membrance focus of dystopy also carrying out vascularization in EM patient's body in uterus, and high-caliber GREM1 may play therein and promote angiopoietic effect.

Five. conclusion:

1. all can detect in endometriosis patient and non-endometriosis patient's the serum and obtain GREM1.

2. the level of the GREM1 in proliferative stage of endometrium dystopy disease patients serum is significantly higher than non-endometriosis patient.

Embodiment 3: a kind of kit that detects endometriosis

One. form:

Detect the enzyme linked immunological kit of endometriosis, be made up of following reagent, 2～8 ℃ keep in Dark Place:

1.48 or 96 hole polystyrene plates;

2.GREM1 mouse monoclonal antibody (H00026585-M07), Abnova company;

3.GREM1 rabbit polyclonal antibody (sc-28873), santa cruz company;

4. enzyme labelled antibody: horseradish peroxidase-labeled goat anti-rabbit igg, santa cruz company;

5.GREM1 standard items: with the PBS dilution, concentration is 0ppb, 100ppb, 200ppb, 400ppb, 800ppb and 1200ppb; (recombinant mouse GREM1,956-GR/CF), R﹠amp; D company;

6. coating buffer: pH 7.2, concentration are the phosphate buffer PBS of 0.02mol/L;

7.10 doubly concentrated cleaning solution PBST:pH 7.4, concentration 1.5mol/L are with KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, NaCl 8.0g, KCl 0.2g and TWeen-200.5ml are dissolved in 100ml distilled water; Use preceding water or deionized water to be diluted to final concentration for 10 times and be 0.15mol/L;

8. confining liquid: 5% skimmed milk is dissolved in 100ml cleansing solution PBST with the 5g skimmed milk power;

9. antibody diluent: 0.5% skimmed milk is dissolved in 100ml cleansing solution PBST with the 0.5g skimmed milk power;

10. substrate colour developing liquid: tetramethyl benzidine 10mg/5ml absolute ethyl alcohol 0.5ml, 0.75%H ₂O ₂32 μ l, pH5.5 substrate buffer solution 10ml, wherein the composition of substrate buffer solution is: 0.1M citric acid (19.2g/L) 24.3ml, 0.2M Na ₂HPO ₄12H ₂O (71.7g/L) 25.7ml and 50ml distilled water;

11. stop buffer: 2M H2SO4, distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml.

Two. using method:

1. kit is opened, equilibrium at room temperature 30 minutes; Concentrated cleaning solution is with distilled water or 10 times of dilutions of deionized water;

2. lath is fixed on the grillage, remaining lath seals and puts into sealing bag and preserve with adhesive sticker;

3. bag quilt: with coating buffer dilution GREM1 monoclonal antibody to protein content is 1～10 μ g/ml, adds 100ul in each reacting hole of polystyrene board, puts 4 ℃ and spends the night; Discard solution in the hole next day,, pat dry with cleansing solution PBST washing 1～3 time;

3. sealing: every hole adds confining liquid 200ul, and 37 ℃ were sealed 2 hours, with lavation buffer solution PBST washing 1～3 time, discards solution in the hole, pats dry;

4. application of sample: every hole adds 50ul sample to be checked respectively and is the GREM1 polyclonal antibody 50ul of 0.2ug/ml with the final concentration of antibody diluent dilution; In blank well, add simultaneously the standard items 50ul of each concentration respectively and be that the GREM1 polyclonal antibody 50ul of 0.2ug/ml is used to make typical curve with the final concentration of antibody diluent dilution; Hatch 30min jointly for 37 ℃,, discard solution in the hole, pat dry with lavation buffer solution PBST washing 3～5 times;

5. add enzyme labelled antibody: every hole adds the enzyme labelled antibody 100ul with antibody diluent dilution in 1: 3000, hatches behind the 30min PBST washing 5 times for 37 ℃, discards solution in the hole, pats dry;

5. chromogenic reaction: every hole adds substrate colour developing liquid 100ul, colour developing 15min;

6. cessation reaction: every hole adds stop buffer 50ul, cessation reaction;

7. the result judges: with microplate reader 450nm reading result of determination, measure the OD value of each reacting hole with zeroing back, blank hole behind the mixing; Standard items absorbance with variable concentrations is that horizontal ordinate, GREM1 concentration are that ordinate carries out linear fit, gets equation of linear regression and typical curve, according to protein content in regression equation or the typical curve calculation sample.When protein content 〉=700ng/ml, infer and suffer from endometriosis.

Three. points for attention:

The present invention adopts not diluted serum to detect, and consumption is 50 μ l.Serum does not use microbiological contamination, piarhemia or haemolysis sample.Collect serum according to standard method, the room temperature preservation sample surpasses 8 hours, if experiment was carried out later at 8 hours, need then be kept at-20 ℃ 1 week as preserving to surpass with sample retention at 2～10 ℃.

Claims (10)

1. a method that detects endometriosis is characterized in that: by measuring the expression of GREM1 albumen in experimenter's endometrial tissue or the serum, judge whether to suffer from endometriosis.

2. a kind of method that detects endometriosis according to claim 1, it is characterized in that: described criterion is: during serum albumin content 〉=700ng/ml, be speculated as and suffer from endometriosis.

3. a kind of method that detects endometriosis according to claim 1 is characterized in that: the method for described detection endometriosis can be Western Blot method, chemoluminescence method, euzymelinked immunosorbent assay (ELISA) or PCR method.

4. according to the described a kind of method that detects endometriosis of claim 3, it is characterized in that: described method is an euzymelinked immunosorbent assay (ELISA), and step is as follows:

(1) bag quilt: with the phosphate buffer of coating buffer 0.02mol/L pH 7.2 the GREM1 monoclonal antibody being diluted to protein content is 1～10 μ g/ml, adds 100ul in each reacting hole of polystyrene board, puts 4 ℃ and spends the night; Discard solution in the hole next day,, pat dry with cleansing solution PBST washing 1～3 time;

(2) sealing: add 5% skimmed milk 200ul, 37 ℃ were sealed 2 hours, with lavation buffer solution PBST washing 1～3 time, discarded solution in the hole, patted dry;

(3) application of sample: every hole adding 50ul sample to be checked and the final concentration that dilutes with 0.5% skimmed milk are the GREM1 polyclonal antibody 50ul of 0.2ug/ml, hatch 30min jointly for 37 ℃, with lavation buffer solution PBST washing 3～5 times, discard solution in the hole, pat dry; Typical curve: every hole adds the 50ul standard items, concentration is 0ppb, 100ppb, 200ppb, 400ppb, 800ppb and 1200ppb, with the final concentration with 0.5% skimmed milk dilution be the GREM1 polyclonal antibody 50ul of 0.2ug/ml, hatch 30min jointly for 37 ℃, with lavation buffer solution PBST washing 3～5 times, discard solution in the hole, pat dry;

(4) add enzyme labelled antibody: every hole adds the HRP-goat anti-rabbit igg 100ul with the dilution in 1: 3000 of 0.5% skimmed milk, 37 ℃ hatch 30min after PBST wash 5 times, discard solution in the hole, pat dry;

(5) chromogenic reaction: every hole adds TMB (tetramethyl benzidine) and uses liquid 100ul, colour developing 15min;

(6) cessation reaction: every hole adds 50ul 2mol/L H ₂SO ₄Cessation reaction;

(7) result judges: on the ELISA detector, in the 450nm place, measure the OD value of each reacting hole with zeroing back, blank hole; Standard items absorbance with variable concentrations is that horizontal ordinate, GREM1 concentration are that ordinate carries out linear fit, gets equation of linear regression, according to protein content in the regression equation working sample.

5. an enzyme linked immunological kit that detects endometriosis is made up of following reagent, and 2～8 ℃ keep in Dark Place:

(1) 48 or 96 hole polystyrene plates;

(2) GREM1 monoclonal antibody;

(3) GREM1 polyclonal antibody;

(4) enzyme labelled antibody;

(5) GREM1 standard items;

(6) coating buffer: pH 7.2, concentration are the phosphate buffer PBS of 0.02mol/L;

(7) 10 times of concentrated cleaning solution PBST:pH 7.4, concentration 1.5mol/L are with KH ₂PO ₄0.2g, Na ₂HPO ₄12H ₂O 2.9g, NaCl 8.0g, KCl 0.2g and TWeen-200.5ml are dissolved in 100ml distilled water; Use preceding water or deionized water to be diluted to final concentration for 10 times and be 0.15mol/L;

(8) confining liquid: 5% skimmed milk is dissolved in 100ml cleansing solution PBST with the 5g skimmed milk power;

(9) antibody diluent: 0.5% skimmed milk is dissolved in 100ml cleansing solution PBST with the 0.5g skimmed milk power;

(10) substrate colour developing liquid: tetramethyl benzidine 10mg/5ml absolute ethyl alcohol 0.5ml, 0.75%H ₂O ₂32 μ l, pH5.5 substrate buffer solution 10ml, wherein the composition of substrate buffer solution is: 0.1M citric acid (19.2g/L) 24.3ml, 0.2M Na ₂HPO ₄12H ₂O (71.7g/L) 25.7ml and 50ml distilled water;

(11) stop buffer: 2M H2SO4, distilled water 178.3ml dropwise adds the concentrated sulphuric acid (98%) 21.7ml.

6. a kind of enzyme linked immunological kit that detects endometriosis according to claim 5 is characterized in that: described (2) GREM1 monoclonal antibody is the GREM1 mouse monoclonal antibody, Abnova company.

7. a kind of enzyme linked immunological kit that detects endometriosis according to claim 5 is characterized in that: described (3) GREM1 polyclonal antibody is the GREM1 rabbit polyclonal antibody, santa cruz company.

8. a kind of enzyme linked immunological kit that detects endometriosis according to claim 5 is characterized in that: described (4) enzyme labelled antibody is the horseradish peroxidase-labeled goat anti-rabbit igg, santa cruz company.

9. a kind of enzyme linked immunological kit that detects endometriosis according to claim 5 is characterized in that: described (5) GREM1 standard items are recombinant mouse GREM1, R﹠amp; D company.

10. a kind of enzyme linked immunological kit that detects endometriosis according to claim 5 is characterized in that: the concentration of described (5) GREM1 standard items is 0ppb, 100ppb, 200ppb, 400ppb, 800ppb and 1200ppb.

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