CN102393458A - Diagnostic reagent for serum of early breast cancer and detection method thereof - Google Patents
Diagnostic reagent for serum of early breast cancer and detection method thereof Download PDFInfo
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Abstract
The invention discloses a diagnostic reagent for serum of early breast cancer and a detection method thereof. A ferritin heavy chain 1 (FTH1) or an FTH1 united heterogeneous nuclear ribonucleoprotein F (hnRNPF) is adopted by the diagnostic reagent to serve as a tumor marker, thus, the diagnostic reagent is capable of detecting an FTH1 antibody in the serum to be detected, or the corresponding antibody of the FTH1 and the hnRNPF, and is used for early diagnosis of the breast cancer or postoperative curative effect monitoring. The reagent disclosed by the invention is verified through the application of the serum of patients with the breast cancer, breast benign tumor and other kinds of cancer (not including the breast cancer) and normal people. The diagnostic reagent for the serum of the early breast cancer is used for the early diagnosis and differential diagnosis of the breast cancer, aims to improve the sensitivity, the specificity and the accuracy of the early diagnosis of the breast cancer and is more beneficial to the diagnosis and the treatment of the patients with the breast cancer, thereby, the problem that the traditional tumor marker of the breast cancer is not ideal is solved, and the goal of improving the sensitivity and the accuracy of the early diagnosis of the breast cancer is achieved.
Description
Technical field
The present invention relates to biological technical field, particularly relate to a kind of early-stage breast cancer serodiagnosis reagent and detection method thereof.
Background technology
Breast cancer is the modal malignant tumour of women.Research shows that breast cancer incidence accounts for 31% of gynecological tumor, and mortality ratio occupies second in tumour, and breast cancer incidence has rising trend in recent years, has become the primary malignant tumour that threatens China's women's health.Though breast cancer does not have special preventive measure at present, but it belongs to a kind of tumour of early stage Yi Zhiyi control, and as if ability early detection, early diagnosis, early treatment, the prognosis situation is generally relatively good.Clinically, the treatment curative effect that part breast cancer is desirable because of early detection has obtained, but most of patient is because of finding the later best occasion for the treatment of having missed.Therefore, in current and quite a long time in the future, strive for that early diagnosis will be an elementary tactics of breast cancer control.
The mode of diagnosis early-stage breast cancer; In diagnosis mainly is to check oneself unusual (check oneself like mammary gland etc.) or hospital's health check-up (as adopting the photography of breast molybdenum target X line, mammary gland colored multispectral rein in ultrasonic examination, breast duct endoscopy, breast duct lavation, CT and magnetic resonance etc. aspect the discovery; If have lump or the abscess that can't distinguish can carry out the cytology examination by centesis), aspect making a definite diagnosis, then be the pathological tissue inspection.But in clinical, there is following defective: check oneself unusual or hospital's health check-up one, at present; Aspect the early stage property prompting of diagnosis, still show and lag behind; Particularly for those because lump is less, or soak into light, become expansive growth, show as smooth, movable, clear border; With the early-stage breast cancer patient of the difficult difference of benign tumour, loss is higher, perhaps when diagnosis is found, has spent best early treatment opportunity; Two, owing to relate to aspect problem such as Social Culture ethics; Some young woman ignore or the generation resisting psychology for breast examination easily; Above-mentioned hospital's health check-up method is more limited to the diagnosis capability that is in the patient with breast cancer of commitment, particularly young patient; Though three, pathological examination is " goldstandard " that tumour is made a definite diagnosis, non-patient with operation is done the pathology inspection and is had the reasons such as tumour cell diffusion risk due to drawing materials, therefore not by widespread usage.
At present; Along with the development of technology,, do the inspection of peripheral blood breast cancer index of correlation mostly for the health check-up and the person of not making a definite diagnosis with regard to the breast cancer aspect; Its advantage is simple and feasible; But still have following shortcoming: one, existing index expression in early days is micro-, and susceptibility and specificity are low, so recall rate is on the low side; Two, existing index is unstable relatively in clinical diagnosis, makes the part patient miss the early diagnosis chance.In addition, supervision of the curative effect of postoperative patient and relapse diagnosis exist to lack the problem that desirable peripheral blood detects index too.
Therefore, find desirable early diagnosis index, its meaning is very great.At present at home and abroad, the current research emphasis in this area has following four aspects:
1. full genome aspect screening New Set
In the existing serological index, breast cancer diagnosis is worth big few, lacks the early diagnosis valuable index especially.Lack desirable diagnosis index, reason possibly be to fail to screen index of correlation comprehensively; The detection of full genomic expression in recent years is existing than quantum jump; Can carry out detections such as display technique of bacteriophage, full genome chip technology; Therefore both at home and abroad begun to screen that all kinds of tumours are new, valuable index from full genome aspect; This scheme can be found new diagnosis index, so become one of research focus.
The exploitation of 2. many index associating diagnostic models
Markers in diagnosis is worth another reason that does not reach re-set target, possibly be that the index difference that tumour is dissimilar, different times is expressed is relevant, and therefore expectation is unrealistic with the diagnosis problem that single index solves each stage.Existing many both at home and abroad index associating Studies on Diagnosis reports, this seminar once unites diagnosis to 12 kinds of common counters to be studied, and obtains better effects.Many indexs are united diagnosis, can improve the accuracy of diagnosis.
3. seek the index that can reflect the tumour early lesion in the peripheral blood
Be the present stage of main means examination tumour with invention Peripheral blood examination, whether the peripheral blood index is desirable, determining the quality of early diagnosis effect.As far as most tumors, still lack desirable diagnosis index at present.Tomour specific albumen is expressed in lesion tissue the earliest, but this being expressed in is micro-in early days, expresses even add lesion tissue, also may not be secreted into peripheral blood, so be necessary from the early expression index, to find out the index that can be secreted in the peripheral blood.In recent years have and attempt to utilize antibody to be index, realize the research of early diagnosis; Its theoretical foundation is, in a single day the focus cell has the change of expressing protein, will produce corresponding antibody through immunological response, and antibody can detect from blood, and existing methodology can detect the antibody of trace in the blood.Remove this, detect micrometastasis tumour cell in the peripheral blood in addition, microRNA is the research of diagnosis index, the gene mutation of dna level etc.
4. the development and application of neomethodology
Methodological sensitivity, specificity are to estimate its good and bad major criterion, and Protein Detection mainly contains protein chip technology, display technique of bacteriophage, enzyme immunological technique etc., and mRNA detects nucleic acid chip technology, the RT-PCR technology etc. of mainly containing.Display technique of bacteriophage is the important means that is used for the New Set screening in recent years.
Display technique of bacteriophage makes up the oncoprotein library; Screen the existing part report of specific index in the serum with this; Zhong Li etc. has reported the result of study in the lung cancer; Obtain better effects, the susceptibility of its product diagnosing non-small cell lung cancer reaches 90 ﹪, specificity reaches 95 ﹪, all exceeds existing any other diagnostic method out and away.The similar research of domestic and foreign literature report mainly also be confined to lung cancer, prostate cancer and oophoroma, and the report aspect breast cancer is actually rare.
See that from development trend the diagnosis index screening of breast cancer etc. mainly is to launch from full genome aspect; Protein is the material form that directly plays physiological action, and is therefore also more valuable than the research of DNA or RNA aspect; Display technique of bacteriophage can be expressed the individual gene of importing, is convenient to the specific aim research of specific protein.Therefore, use phage display and make up full genome albumen library, the protein index that examination is new has become research trend.
In view of this, the inventor is combined in the experience of working for many years in this field as specializing in the doctor that breast cancer diagnosis detects, and the problems referred to above and cutting edge technology are carried out long-term follow research, and this case produces thus.
Summary of the invention
The objective of the invention is to overcome the existing undesirable problem of tumor markers of breast cancer; Be intended to improve susceptibility, specificity and the accuracy rate of early diagnosing mammary cancer, utilize a species specificity, the diagnosis marker preparation diagnosis good stability that susceptibility is strong, the diagnostic reagent that is used for early diagnosing mammary cancer or postoperative curative effect monitoring easy to detect.The present invention is used for the early diagnosis and the antidiastole of breast cancer, more helps patient with breast cancer's diagnosis and treatment, reach improve mammary gland early diagnosis susceptibility and with the target of accuracy.
Another object of the present invention is to provide a kind of method of carrying out early diagnosing mammary cancer or postoperative curative effect monitoring.
For realizing above-mentioned purpose, technical scheme of the present invention is following:
A kind of early-stage breast cancer serodiagnosis reagent; Said diagnostic reagent is with FTH1, and perhaps FTH1 associating hnRNPF is a diagnosis marker, can detect FTH1 antibody in the test serum of separation; Perhaps the corresponding antibody of FTH1 and hnRNPF is used for early diagnosing mammary cancer or postoperative curative effect monitoring.
A kind of early-stage breast cancer serodiagnosis reagent; Comprise the FTH1 elisa plate; The FTH1 antigen embedding ELISA Plate that the FTH1 elisa plate is buied by the FTH1 antigen of using breast cancer T7 bacteriophage specifically expressing or commercialization earlier, 4 ℃ are spent the night, and the 5%BSA/PBS room temperature sealing of washing back formed in 2 hours; Also comprise goat anti human IgG, developer A, B and the stop buffer that can cooperate the HRP coupling that the FTH1 elisa plate detects test serum; The FTH1 elisa plate can read the OD value in 450 nm wavelength by ELIASA, with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole, judge ratio >=2.0 according to the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control; Be judged as the positive, ratio＜2.0 are judged as feminine gender; Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
Further; A kind of early-stage breast cancer serodiagnosis reagent; Also comprise the hnRNPF elisa plate; The hnRNPF antigen embedding ELISA Plate that the hnRNPF elisa plate is buied by the hnRNPF antigen of using breast cancer T7 bacteriophage specifically expressing or commercialization earlier, 4 ℃ are spent the night, and the 5%BSA/PBS room temperature sealing of washing back formed in 2 hours; Also comprise goat anti human IgG, developer A, B and the stop buffer that can cooperate the HRP coupling that the hnRNPF elisa plate detects test serum; The hnRNPF elisa plate can read the OD value in 450 nm wavelength by ELIASA, with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole; Ratio according to the average OD value of hnRNPF elisa plate test serum OD value/negative control; Ratio in conjunction with the average OD value of FTH1 elisa plate test serum OD value/negative control; Judge that negative control OD value is lower than 0.05 and does 0.05 calculating, be higher than 0.05 by actual OD value calculating.
A kind of method of carrying out early diagnosing mammary cancer or postoperative curative effect monitoring may further comprise the steps:
(1) dosing: use breast cancer T7 bacteriophage specifically expressing FTH1 antigen and process T7 phagocytosis body fluid, perhaps directly the FTH1 antigen liquid is bought in commercialization, and perhaps directly commercialization is bought FTH1 antigen powder and processed the FTH1 antigen liquid;
(2) preparation of FTH1 elisa plate: T7 phagocytosis body fluid or FTH1 antigen liquid are encapsulated in ELISA Plate, and 4 ℃ are spent the night; After washing, 5%BSA/PBS room temperature sealing 2 hours;
(3) specimen collection and processing: take out peripheral blood to be measured, separate test serum;
(4) detection of FTH1 elisa plate: the test serum incubated at room of adding 1:100 dilution in every hole of ELISA Plate 1 hour; The washing back adds the goat anti human IgG of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; The washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃;
(5) read plate: 450 nm wavelength read the OD value under ELIASA;
(6) according to the feminine gender of OD value, positive judged result: with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole, judge ratio >=2.0 according to the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control; Be judged as the positive, ratio＜2.0 are judged as feminine gender; Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
Further, a kind of method of carrying out early diagnosing mammary cancer or postoperative curative effect monitoring, further comprising the steps of:
(1) dosing: use breast cancer T7 bacteriophage specifically expressing hnRNPF antigen and process T7 phagocytosis body fluid, perhaps directly the hnRNPF antigen liquid is bought in commercialization, and perhaps directly commercialization is bought hnRNPF antigen powder and processed the hnRNPF antigen liquid;
(2) preparation of hnRNPF elisa plate: T7 phagocytosis body fluid or hnRNPF antigen liquid are encapsulated in ELISA Plate, and 4 ℃ are spent the night; After washing, 5%BSA/PBS room temperature sealing 2 hours;
(3) specimen collection and processing: take out peripheral blood to be measured, separate test serum;
(4) detection of hnRNPF elisa plate: the test serum incubated at room of adding 1:100 dilution in every hole of ELISA Plate 1 hour; The washing back adds the goat anti human IgG of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; The washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃;
(5) read plate: 450 nm wavelength read the OD value under ELIASA;
(6) according to the feminine gender of OD value, positive judged result: with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole; Ratio according to the average OD value of hnRNPF elisa plate test serum OD value/negative control; Ratio in conjunction with the average OD value of FTH1 elisa plate test serum OD value/negative control; Judge that negative control OD value is lower than 0.05 and does 0.05 calculating, be higher than 0.05 by actual OD value calculating.
Further; In said (6) step according to the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control; Ratio in conjunction with the average OD value of FTH1 elisa plate test serum OD value/negative control is judged; Be meant the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control or the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control, arbitrary ratio >=2.0 promptly are judged as the positive.Thereby obviously improve the breast cancer diagnosis effect.
The theoretical foundation of this method: the existing (Hou Zhenjiang that discovers; Zhang Zongying. breast cancer tumour Progress of Study about Marker [J]. medical science summary 2008; 14 (2): 224-226) commitment, the symptom in multiple cancer do not occur as yet; Even pathology detect when all can not find to change, and the antibody to cancer has just been arranged in cancer patient's blood.Because antibody more early appears in the serum, adopts this technical scheme can reach the effect of more early diagnosis.
This research discards the bacteriophage of combination at first with the antibodies in phage library and the normal control group serum, promptly rejects non-specific bacteriophage.Remaining bacteriophage again with patient with breast cancer's serum in antibodies, bacteriophage that can specific bond is collected and is increased.This is to take turns positive and negative affine screening, four-wheel so repeatedly, and the bacteriophage that enables to combine with the breast cancer specific antibody will obtain the enrichment of enough degree.
The inventor is through separating and research healthy women serum and patient with breast cancer's serum; Handle through breast cancer T7 phage library; Choose and the high specific of breast cancer height correlation and the special phage clone of susceptibility, again these specificitys clone bacteriophage is done pcr amplification, order-checking, screening comparison in full genomic expression spectrum; Find the tumor markers that can be used for early diagnosing mammary cancer, finally develop the early-stage breast cancer serodiagnosis reagent that to be convenient to clinical practice.
The present invention is used for the early diagnosis and the antidiastole of breast cancer, improves susceptibility, specificity and the accuracy rate of early diagnosing mammary cancer, more helps patient with breast cancer's diagnosis and treatment.Solve the existing undesirable problem of tumor markers of breast cancer with this, reach improve mammary gland early diagnosis susceptibility and with the target of accuracy.
This breast cancer diagnosis mark (hnRNPF:heterogeneous nuclear ribonucleoprotein F; FTH1:Ferritin heavy chain 1) be from full genomic expression spectrum, to screen to obtain; Has maximum comprehensive, promptly maximum representativeness during selection.This breast cancer diagnosis mark (hnRNPF, FTH1) is a breast cancer specificity marker thing through patient with breast cancer's lesion tissue, cancer beside organism, the checking of normal control group.This breast cancer diagnosis mark (hnRNPF, FTH1) is not only at breast cancer focus tissue specificity high expressed; And also synchronous high expressed among the patients serum; Empirical tests, this mark high expressed has the existing higher accuracy of other indexs to the diagnosis of breast cancer in the serum.This breast cancer blood serum designated object is a kind of antibody, and can reach more early stage diagnosis effect like corresponding antibodies in the aforementioned detection serum, and this has diverse theory and effect with the diagnostic antigen mark of reporting and use in the past.Be that the present invention not only screens new breast cancer blood serum designated object, and be to use its antibody a kind of new departure as detected object.
The disclosed breast cancer diagnosis mark of present technique scheme has higher susceptibility, specificity and accuracy rate; Therefore the required serum to be checked of the diagnostic reagent of making in view of the above is considerably less; Add present antibody test technology maturation, can detect its existence under the situation of denier, remedied antigen or other existing diagnosis markers in the difficult detected deficiency of micro-situation; Add that antibody more early appears in the serum, from reaching the purpose of early diagnosis.
The reagent of the present invention's exploitation through the checking that breast cancer, mammary gland benign tumor, other cancers (non-breast cancer) patient and normal human serum are used, is specifically seen the embodiment part.
Below in conjunction with accompanying drawing the present invention is explained further details.
Description of drawings
Fig. 1 eluriates the process of breast cancer specific expression protein for bacteriophage.
Fig. 2 is for inserting the phagocytosis of breast cancer specific gene.
Fig. 3 is the breast cancer phage display peptide library.A is original storehouse, and B is an amplification bank.
Fig. 4 is part bacteriophage pcr amplification product agarose electrophoresis figure.M:DL 2000 Marker; 1～16 at random clone insertion fragment PCR result.
Fig. 5 is the RT-PCR part amplified production electrophoretogram of hnRNPF mRNA and FTH1 mRNA.A:hnRNPF mRNA part amplified production electrophoretogram, B:FTH1 mRNA part amplified production electrophoretogram; M:DL2000 mark, 1,2,3,4 are breast cancer focus tissue, and 5,6 is mammary gland cancer beside organism, and 7,8 are the mammary gland benign tumor tissue, 9 negative contrasts: replace substrate with deionized water.
Fig. 6 is hnRNPF mRNA and the expression of FTH1 mRNA in different tissues.
Fig. 7 is two indexes serum and variance analysis of healthy women expression of serum before patient with breast cancer's art.
Fig. 8 be the expression ratio of two indexes in serum and cancer kitchen range tissue.
Fig. 9 is the ROC curve of hnRNPF, FTH and CA15-3 individual event or associating diagnosing mammary cancer.
Figure 10 is ROC TG-AUC and 95% credibility interval.
Figure 11 is the value of two indexes early diagnosis breast cancer.
Figure 12 is the relation of two indexes and breast cancer clinical stages.
Figure 13 is the relation of two indexes and breast cancer pathology somatotype.
Figure 14 is the relation of two indexes and Metastasis in Breast Cancer.
Figure 15 detects ELISA ELISA Plate photo for the hnRNPF antibody serum.
Figure 16 detects ELISA ELISA Plate photo for the FTH1 antibody serum.
Embodiment
One, the used sample of the present invention source, reagent consumptive material and key instrument
1, sample source
Agree through me and family members thereof; The breast cancer flesh tissue is totally 40 examples; Be taken from the patient of hospital's mammary gland and thyroid gland surgery in May, 2009 to 2010 year operation in May, focus and cancer beside organism transfer to liquid nitrogen container rapidly after downcutting, and 80 ℃ of ultra low temperature freezers of – are preserved subsequent use; Patient with breast cancer's serum specimen is totally 273 examples, divides into groups according to clinical stages, pathology somatotype and transfer; Getting the female patient serum sample that normal women of 54 examples and 40 examples suffer from other tumours simultaneously preserves subsequent use as 80 ℃ of ultra low temperature freezers of control group ， –.Concrete clinical data is seen table 1.
Table 1 patient with breast cancer, his cancer patient and normal control crowd's main clinical and pathological data
| Project | Breast cancer | Other cancers | The normal control group |
| Number | 273 | 40 | 54 |
| Age | ? | ? | ? |
| The range of age (median) | 23～74( 47) | 26～81( 59) | 28～59( 41) |
| (non-early stage) breast cancer in early days | ? | ? | ? |
| Early-stage breast cancer # | 33(12.1) | ? | ? |
| Non-early-stage breast cancer | 187( 68.5) | ? | ? |
| Other ★ | 53( 19.4) | ? | ? |
| Histological type * [number (%)] | ? | ? | ? |
| IDC | 189( 69.2) | ? | ? |
| ILC | 26( 9.5) | ? | ? |
| LCIS | 17( 6.2) | ? | ? |
| Other ★ | 41( 15.1) | ? | ? |
| Clinical stages [number (%)] | ? | ? | ? |
| 0～I phase | 58( 21.2) | ? | ? |
| The II phase | 73( 26.7) | ? | ? |
| The III phase | 56( 20.5) | ? | ? |
| The IV phase | 27( 9.9) | ? | ? |
| Other ★ | 59( 21.7) | ? | ? |
| Axillary lymphatic metastasis [number (%)] | ? | ? | ? |
| Do not see transfer | 78 ( 28.6) | ? | ? |
| Shift | 116( 42.5) | ? | ? |
| Other ★ | 79(28.9) | ? | ? |
Annotate: early-stage breast cancer is according to the standard arrangement for early-stage breast cancer such as Li Xinghui.The breast carcinoma tissue form is comparatively complicated, numerous types, and often in same cancerous tissue, even can have two or more types to exist simultaneously in same section.International, domestic at present breast cancer pathological classification, ununified yet in practical application.The listed pathological classification of this group testing data is listed to be 273 routine breast cancer pathology classification front threes.
★Represent indeterminate person.
2, reagent consumptive material
Breast cancer cDNA T7 phage library (breast cancer cDNA T7 phage library) is available from Novagen company; Trizol reagent (Trizol reagent) is available from Invitrogen company; T7 Select UP Primer:5 '-AACCCCTCAAGACCCGTTTA-3 ', T7 Select DOWN Primer:5 '-GGAGCTGTCGTATTCCAGTC-3 ' is available from Novagen company; PARISTM Kit purchases the AMBION company in the U.S.; The IgG of the anti-human albumin antibody of rabbit, HRP-goat-anti rabbit, protein A/G plus-Agarose magnetic bead are purchased the company in SANTA CRUZ.Other reagent such as tables 2.
Other reagent of table 2
| Reagent name | Manufacturer |
| Sodium chloride (NaCl) | Reagent four factories of Shanghai chemical reagent company limited |
| Potassium chloride (KCl) | Shanghai chemical reagents corporation of Chinese Medicine group |
| ADSP (Na2HPO4) | Shanghai chemical reagents corporation of Chinese Medicine group |
| Anhydrous potassium dihydrogenphosphate (KH2PO4) | Shanghai chemical reagents corporation of Chinese Medicine group |
| Methyl alcohol (CH3OH) | Shanghai chemical reagents corporation of Chinese Medicine group |
| Ethylenediamine tetraacetic acid (EDTA) | Shanghai chemical reagents corporation of Chinese Medicine group |
| Ethanol (CH3CH2OH) | Shanghai development chemical industry one factory |
| Bromination is ingot (EB) | Amresco |
| Bovine serum albumin(BSA) (BSA) | Worker's bioengineering company limited is given birth in Shanghai |
| Tween-20 (Tween-20) | Shanghai chemical reagents corporation of Chinese Medicine group |
| 30% polyacrylamide (Polyacrylamid) | Worker's bioengineering company limited is given birth in Shanghai |
| Ammonium persulfate (Ammonium Presulfate, AP) | Photo bio Science and Technology Ltd. is won in Shanghai |
| Tetramethylethylenediamine (TEMED) | Worker's bioengineering company limited is given birth in Shanghai |
| Agar powder (Agar) | Shanghai chemical reagents corporation of Chinese Medicine group |
| Yeast extract (Yeast Extract) | OXOID |
| Tryptone (Tryptone) | OXOID |
| Agarose (Agarose) | Purple special Science and Technology Ltd. is liked in Shanghai |
| Ampicillin (Ampicillin, Amp) | Amresco |
| Glycerine (glycerol) | Amresco |
| Lauryl sodium sulfate (SDS) | Amresco |
| T7 tail antibody | Novagen |
| The goat anti human IgG that the HRP coupling joins | KPL |
| Deoxy-ribonucleoside triphosphate (dNTP) | TIANGEN Biotech (Beijing) Co., Ltd. |
| 10 * taq enzyme buffer liquid | TIANGEN Biotech (Beijing) Co., Ltd. |
| Developer A and B, stop buffer | Shanghai Kehua Bio-technology Co., Ltd |
| 25 * washing lotion | Shanghai Kehua Bio-technology Co., Ltd |
| Protein A/G Plus-sepharose 4B | Bio basic Inc |
3, key instrument
ELIASA (model: MK-3, the place of production: China); Pcr amplification appearance (model: Bio-RadS1000, the place of production: the U.S.); Ultra low temperature freezer (model: 705THERMOFORMA, the place of production: the U.S.); High speed freezing centrifuge (model: FRESCO, the place of production: the U.S.); A complete set of electrophoresis and protein purification system (model: ProteomeWorksTMSystem, the place of production: the U.S.); Biohazard Safety Equipment (model: HFSAFE900, the place of production: the Hong-Kong); Ultraviolet gel imaging appearance (model: ProteomeWorksTMSystem, the place of production: the U.S.); Gel electrophoresis imaging system (model: ChemiDocTM XRS+, the place of production: the U.S.).The used abbreviation of the present invention, symbol inventory and nomenclature are seen table 3.
Table 3 abbreviation, symbol inventory, nomenclature
| Abbreviation | Full Name in English | Chinese full name |
| RNA | Ribonucleic Acid | RNA. |
| mRNA | messenger RNA(ribonucleic acid) | Messenger RNA |
| DNA | deoxyribonucleic acid | Ribodesose nuclear |
| cDNA | complementary deoxyribonucleic acid | Complementary DNA (cDNA) |
| dNTP | deoxy-ribonucleoside triphosphate | The triphosphoric acid DNA |
| LB | Luria-Bertani medium | The LB nutrient culture media |
| bp | base pair | Base-pair |
| EB | ethidium bromide | Ethidium bromide |
| E.coli | Escherichia coil | Escherichia coli |
| IgG | immunoglobulin G | Immunoglobulin G |
| OD | optical density | Optical density |
| SDS | sodium dodecyl sulphate | Lauryl sodium sulfate |
| EDTA | ethylenediaminotetraacetic acid | Ethylenediamine tetraacetic acid |
| PBS | Phosphate buffered saline | Phosphate buffered saline (PBS) |
| rpm | revolutions per minute | Rotations per minute |
| pfu | plaque forming unit | Plaque-forming unit |
| Tris | tris(hydroxymethyl)aminomethane | Trishydroxymethylaminomethane |
| PCR | polymerase chain reaction | Polymerase chain reaction |
| DEPC | diethyl pyrocarbonate | DEPC |
| BSA | bovine serum albumin | Bovine serum albumin(BSA) |
| ELISA | enzyme-labeled immunosorbent assay | Enzyme linked immunosorbent assay (ELISA) |
| HRP | horseradish peroxidase | Horseradish peroxidase |
| KD | kilodalton | Kilodalton |
| TEMED | tetramethylethylenediamine | Tetramethylethylenediamine |
| APS | Ammonium Presulfate | Ammonium persulfate |
| hnRNP | heterogeneous nuclear ribonucleoprotein | Heterogeneity ribonucleoprotein in the nuclear |
Two, hnRNPF, FTH1 verify as the specificity of markers for breast cancer
1, specimen collection
As aforementioned, 40 routine patient with breast cancers agree through me and family members thereof, gather flesh tissue 0.1-0.5g in preceding peripheral blood 5 ml of art and the art respectively, and focus and cancer beside organism transfer to liquid nitrogen container rapidly after downcutting, and 80 ℃ of ultra low temperature freezers of Ran Hou – are preserved subsequent use; Get the normal women's peripheral blood of 54 examples simultaneously as control group, 3000 g/ minutes centrifugal 5 minutes, upper serum is transferred to 80 ℃ of ultra low temperature freezers of EP Guan Zhong – preserves subsequent use.
2, phage display and elutriation
(1) amplification of phage display library
Get behind the BLT5615 bacterium line LB/AMP plate in 37 ℃ of incubations, picking list bacterium colony is in fresh TB fluid nutrient medium, and 37 ℃ of 220 rpm shook about 4 hours; Surveying the OD value is 0.8～1.0 o'clock; From the breast cancer phage peptide library that builds, get l0 μ l phagocytosis body fluid, 250 μ l BLT5615 bacterium liquid, 3 ml preheatings and be incubated 55 ℃ top agar; Pour into after shaking up rapidly to the LB/AMP plate of 37 ℃ of insulations; Be placed upside down in 37 ℃ of constant temperature ovens after cooling and hatched 4 hours, the counting plaque.20 plaques of picking are put into the EP pipe that has added 100 μ l l0mM EDTA (pH 7.5) respectively at random, and 65 ℃ were heated 10 minutes, and 14 000 rpm/ minutes centrifugal 3 minutes.Supernatant promptly contains the template of phage DNA, carries out pcr amplification.1.5% agarose gel electrophoresis is observed and is inserted clip size.The T7 upstream and downstream primer approximately 130bp fragment that when not having exogenous segment to insert, can increase on the bacteriophage both arms.Counting has the sample that inserts fragment, calculates recombination fraction.
Table 4LB culture medium prescription (1L)
Table 5TB nutrient solution prescription (1L)
Table 6 kaliumphosphate buffer prescription (1L)
Table 7 top agar prescription (100ml)
(2) elutriation, amplification and the library titer determination of the special antigen bacteriophage of expression breast cancer
The sepharose 4B of getting 10 μ l protein-A/G is in 1.5 ml Eppendorf pipe, and l * PBS of pH 7.4 washes 2 times, 4 ℃ of sealings of 1%BSA l hour.Sepharose 4B respectively with 15 μ l patient with breast cancers and 4 ℃ of incubated overnight of normal human serum.After the 1 * PBS washing 3 times, dissolve the antibody that enrichment is arrived with 1 * PBS of l0 μ l.5 pipe breast cancer antibody and 5 pipe normal person antibody are merged into a pipe separately.Get phage library amplification liquid 20 μ l,, collect and do not combine supernatant earlier with normal person's antibody incubation l h of 20 μ l, supernatant again with 20 μ l patient with breast cancer antibodies, keep being attached to the bacteriophage on the sepharose 4B, and be eluted to 100 μ l.Bacteriophage is eluriated process shown in the process that Fig. 1 bacteriophage is eluriated the breast cancer specific expression protein.
Amplification procedure in the Escherichia coli: 1. with 1% inoculum concentration with Escherichia coli BLT 5615 bacterial classification inoculations in LB fluid nutrient medium (containing final concentration is 50 μ g/mL Carb), 37 ℃ of 220 rpm incubated overnight; 2. get the above-mentioned bacterium liquid of 100 μ L in 5 mL LB fluid nutrient mediums (containing final concentration is 50 μ g/mL Carb), 37 ℃, 220 rpm are cultured to OD
600=0.5, add 0.8 M IPTG (final concentration is 1 mmol/L), continued to shake 30 minutes, 4 ℃ then, subsequent use.3. the breast cancer library suspension of getting after 2 μ L eluriate mixes with the subsequent use BLT 5615 of 1mL, gets the library and mixes with 10 mL upper strata LB nutrient culture media with potpourri 1 mL of bacterium liquid, is laid on then on the LB solid plate (50 μ g/mL Carb).4. after treating upper strata nutrient culture media full solidification, be inverted in 37 ℃ of incubators and cultivate, up to plaque clearly occurring.5. get 10 mL plaque extracts on the flat board that grows clear plaque, 4 ℃, spend the night.6. collect the bacteriophage that elution goes out, all collection liquid as in one 15 mL centrifuge tube, is added 0.5 mL chloroform mixing gently, centrifugal 5 minutes of 5000 rpm collect supernatant in a new centrifuge tube, deposit for 4 ℃, as the biological library of eluriating of next round.
More than two processes be to take turns screening.4 take turns so repeatedly, isolate breast cancer immunogenicity phage clone.
Table 8 plaque extract recipe
| Reagent | Consumption |
| 20X M9 salt | 5 |
| 20 |
2 ml |
| 1 M MgSO4 | 0.1 |
| TB | |
| 100 ml |
Eluriate the mensuration of library, back titre: 1. with 1% inoculum concentration with Escherichia coli BLT 5615 bacterial classification inoculations in M9LB fluid nutrient medium (containing final concentration is 50 μ g/mL Carb), 37 ℃ of 220 rpm incubated overnight.2. get the above-mentioned bacterium liquid of 1 mL in 50 mL M9LB fluid nutrient mediums (containing final concentration is 50 μ g/mL Carb), 37 ℃, 220 rpm are cultured to OD
600=1.0, before using, deposit in 4 ℃ of refrigerators, the time can not be above 48 hours.3. gradient dilution is carried out in the library that library after getting that the above-mentioned four-wheel of 1 μ L is biological and eluriating and 10 μ L four-wheels are eluriated after the amplification, dilutability by 10 times from 10
-2Be diluted to 10
-9Begin from high dilution; Each dilutability is respectively got 100 μ L and is mixed with the cultured host bacterium of 250 μ L BLT 5615; In this potpourri, add LB top layer nutrient culture media 3 mL; Be laid on then on the 90 mm LB solid plates (50 μ g/mL Carb), spiral is taped against on the plate so that the top layer nutrient culture media is smooth equably rapidly.4. after treating upper strata nutrient culture media full solidification, 37 ℃ of incubators are inverted and are cultivated, up to plaque clearly occurring.5. each is cloned in the plate count that 10-20 bacterial plaque arranged approximately, calculates its titre.Titre computing formula: titre=each dull and stereotyped number * extension rate * 10 of going up plaque.
Confirming of the amplification of quiding gene and expression breast cancer differential protein bacteriophage
Picking 4 is taken turns back 100 the bacteriophage monoclonals clone of screening and is carried out pcr amplification.Bacteriophage pcr amplification process: 1. get bacteriophage extract 10 μ L, add in the 200 μ L EP pipe; 2. rock the EP pipe gently, 65 ℃ were heated 10 minutes then; 3. cool to room temperature 14000 r/3 minutes centrifuging and taking supernatants then; 4. in the aseptic EP pipe of 200 μ L, add 2 μ L Phage lysate, 5 μ l 10X Buffer with MgCl2,1 μ L T7 Select UP Primer (5 pmol/ μ L), 1 μ L T7 Select DOWN Primer (5 pmol/ μ L), 1 μ l dNTP mix (10 mM each:dATP; DCTP; DGTP, dTTP), 1.25 U DNA polymerase, 38.75 μ L deionized water mix.5. first reacting by heating thing to 80 ℃ adds archaeal dna polymerase again; Carry out following response procedures then: 94 ℃ 50 seconds, 50 ℃ 1 minute, 72 ℃ 1 minute, totally 35 circulations were extended 6 minutes after 72 ℃; 6. agarose gel electrophoresis filtered out the above phage clone of amplified fragments 300bp after pcr amplification was accomplished.
Picking 4 is taken turns the screening back above phage clone of pcr amplified fragment 300bp (sequence with bacteriophage otch two ends is a primer), for expressing breast cancer differential protein bacteriophage, shown in the bacteriophage that Fig. 2 inserts the breast cancer specific gene.
3, order-checking and information science analysis
(1)With the breast cancer specifically expressing bacteriophage of confirming that contains quiding gene, give its PCR product to carry out determining nucleic acid sequence.
Carry out sequence alignment through http://www.ncbi.nlm.nih.gov/blast and find known sequence in full accord or immediate.
Come clear and definite gene location through checking Map figure, if known is known the gene title.
See that whether existing research relevant with cancer if known is consulted document, infer the whether relevant of gene that we obtain with the function of its immediate gene with cancer if the uncorrelated or gene do not studied is then searched those through website such as http://string.embl.de/ and http: // www.chilibot.net//wait.
4, the clinical value of breast cancer specifically expressing index checking
(1) RT-PCR of lesion tissue and cancer beside organism's breast cancer specifically expressing index detects
The extraction of total RNA is with reference to the Trizol operation instructions in breast cancer, cancer beside organism and the mammary gland benign tumor tissue.Total RNA 1 μ g reverse transcription is cDNA, reaction conditions: 30 ℃ 10 minutes, 42 ℃ 30 minutes, 95 ℃ 5 minutes.RT-PCR amplification hnRNPF and FTH1/ β-Actin mRNA; Primer is synthetic by Shanghai Sangon Biotech (Shanghai) Co., Ltd.; Sequence is following: 1. hnRNPF: upstream primer 5 '-CCCTGGTCCTGCT CTGTT-3 '; Downstream primer 5 '-GGCAATGTGATCCCGTTT-3 ', the purpose fragment length is 361bp; 2. FTH1: upstream primer 5 '-TACGCCTCCTACGTTTACC-3 ', downstream primer 5 '-GGCTTTC ACCTGCTCATT-3 ', the purpose fragment length is 317bp; 3. β-Actin: upstream primer 5 '-TTCCTT CTTGGGTATGGAAT-3 ', downstream primer 5 '-GAGCAATGATCTTGATCTTC-3 ', the purpose fragment length is 200 bp.Amplification condition: 95 ℃ of preparatory sex change in 3 minutes, 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 1 minute, 35 circulations; 72 ℃ of extensions in 7 minutes.With distilled water as negative control.Get and carry out 1% Ago-Gel (ethidium bromide 10 mg/ml) electrophoresis after 5 μ l RT-PCR amplified productions and 1 μ l, 6 * Loading buffer liquid mix, computer scanning imaging judged result.Total RNA extract, reverse transcription reaction liquid, reverse transcriptase, RNA enzyme inhibitor are all purchased the precious biotech firm in Dalian.
Reverse transcription reaction process: the 1. following mixed liquor of configuration in Microtube pipe: 1 μ L dNTP Mixture, 1 μ L Oligo dT Prime, 5 μ L Template RNA, add RNase Free dH2O to 10 μ L; 2. be that mixed liquors such as template ribonucleic acid and primer are gathered in the Microtube pipe end after the centrifugal several seconds; 3. in above-mentioned Microtube pipe, prepare following inverse transcription reaction liquid: the above-mentioned mixed liquor of 10 μ L, 4 μ L, 5 * PrimeScriptTMM Buffer, 0.5 μ L RNase Inhibitor, 0.5 μ L PrimeScript
TMRNase, 5 μ L RNase Free dH2O; 4. on the PCR appearance, carry out reverse transcription reaction by following condition: 42 ℃ 30 minutes; 95 ℃ 5 minutes.
The PCR course of reaction: the following mixed liquor of configuration in the Microtube pipe: 5 μ L, 10 * PCR Buffer, 0.5 μ L dNTP Mixture, 0.5 μ L Taq enzyme, 0.5 μ L upper reaches specific PCR primer, 0.5 μ L downstream specific PCR primer, 2 μ L cDNA templates add water to 50 μ L.Temperature of reaction: 94 ℃ 30 seconds, 52～55 ℃ 30 seconds (55 ℃ of β-actin, 52 ℃ of hnRNPF, FTH152 ℃), 72 ℃ 1 minute, totally 35 circulations.
Get after reaction is accomplished and carry out 1% Ago-Gel (ethidium bromide 10 mg/ml) electrophoresis after 5 μ L amplified productions and 1 μ L, 6 * Loading buffer liquid mix.After electrophoresis finishes, use ChemiDoc
TMXRS+ gel electrophoresis imaging system is analyzed.The result judges: sample agarose electrophoresis photo adopts Quantity one analysis software to carry out gray scale scanning, measures the integral optical density (IA) of sample to be tested and confidential reference items β-Actin.The numerical value of sample size representes with the ratio of the IA of purpose fragment IA/ β-Actin, with the integral optical density average of the integral optical density ratio/cancer beside organism of breast cancer (benign tumour) lesion tissue>1.5 be high expressed.
The ELISA of breast cancer specifically expressing indicator antibody detects in the peripheral blood
Utilize the phagocytosis body fluid of expressing the breast cancer candidate markers to prepare elisa plate; Plank layout such as distribution plan; Detection is at patient with breast cancer, other cancer patients and normal control group serum: 1. the T7 phage expression of l:1000 dilution is antigen coated in 96 hole ELISA Plates, and 4 ℃ are spent the night; 2. after washing, 200 μ L 5%BSA/PBS room temperatures were sealed 2 hours; 3. add the bacteriophage sample 100 μ L incubated at room of 1:5 dilution 2 hours; 4. each bacteriophage sample of washing back adds the 5 pipe mixed serum of breast cancer and each 100 μ L of control serum of 1:100 dilution, incubated at room 1 hour; 5. the washing back adds the goat anti human IgG 100 μ L of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; 6. the washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃; 7. 450 nm wavelength read the OD value under ELIASA.Triplicate was all wanted in every hole when the ELISA method detected.
Well distributed when table 9ELISA detected peripheral blood breast cancer specifically expressing index
| B | N | N | N | P | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
| S | S | S | S | S | S | S | S | S | S | S | S |
Annotate: B: blank, N: negative control, P: positive control, S: sample to be measured
Establish 3 holes at every turn and encapsulated the anti-negative control wells in hole of the anti-and sheep anti mouse HRP of bacteriophage, mouse-anti bacteriophage one two, and establish 1 blank hole (not increase serum).Calculate and judged result by following formula: after the blank well zeroing, the average OD value of sample OD value/negative control, ratio >=2.0 are judged as the positive; The average OD value of sample OD value/negative control, ratio＜2.0 are judged as feminine gender.Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
The clinical detection index CA15-3 commonly used of ELISA method testing result and breast cancer makes the positive rate diversity ratio; With normal control group and the negative contrast of other cancer groups; Experimenter's working curve (ROC) figure that makes breast cancer serum breast cancer specifically expressing indicator antibody and CA15-3 comes; Estimate two candidate markers and CA15-3 diagnostic value, and add up according to early diagnosis, clinical stages, histological type and the relations of metastasis of two kinds of candidate markers and breast cancer to breast cancer.
5, statistical method
Adopt the SPSS16.0 statistical software; Measurement data is used
s and is described, and mean relatively adopts the t check between group.Enumeration data use-case number, percentage are described, and relatively adopt Chi-square Test between group.Drawing serum two candidate markers and CA15-3 experimenter's operating characteristic (ROC) curve with SPSS16.0 software, is the excellent diagnostics critical value with the detected value of diagnostic index (sensitivity+specificity) maximum.With
P<0.05 for significant difference is arranged.
6, result
(1) amplification of breast cancer T7 phage display library
Breast cancer T7 Select cDNA phage display library is paved plate and is detected storage capacity after dilution, the original library of T7 phage display cDNA recombinant clone number (Fig. 3 A) after increasing (Fig. 3 B) titre by 1.1 * 10
7Pfu is increased to 6.4 * 10
10Pfu/mL (table 10).
The recombination fraction and the titre of phage peptide library before and after the screening that table 10 four-wheel is affine
| ? | The 1st takes turns enrichment | The 2nd takes turns enrichment | The 3rd takes turns enrichment | The 4th takes turns enrichment |
| Recombination fraction | 70% | 80% | 90% | 95% |
| Titre | 1.1×10 7pfu | 8.5×10 8 | 3.3×10 9 | 6.4×10 10 |
(2) selection of breast cancer specifically expressing bacteriophage behind the pcr amplification
100 plaque amplified productions of picking are at random carried out PCR identify 90% insertion fragment length in the recombinant 300 bp, part agarose electrophoresis result sees Fig. 4 part bacteriophage pcr amplification product agarose electrophoresis figure.M:DL 2000 Marker; 1～16 at random clone insertion fragment PCR result.
The sequencing of breast cancer specific expression gene amplified fragments and information science analysis
Select 84 monoclonal bacteriophage order-checkings of inserting the breast cancer specific gene, the known array in gained gene sequencing result and the GeneBank database is compared analysis.Come clear and definite gene location through the NCBI sequence alignment and through Map figure, known is obtained gene title (seeing table 11), wherein hnRNPF is relevant with cancer with the FTH1 two indexes, and therefore choosing these two kinds of indexs carries out clinical verification as candidate markers.The said gene title all belongs to the general knowledge of this area, is not just giving unnecessary details here.
The gene that table 11 screens
| The sequenced genes label | The sample numbering |
The |
| 1 | 1 | |
| 2 | 12 | |
| 3 | 13 | B 2-M |
| 4 | 17 | |
| 5 | 67 | SLC40A1 |
(4) lesion tissue and the breast cancer specifically expressing index RT-PCR of cancer beside organism detect
Breast cancer focus tissue, cancer beside organism, mammary gland benign tumor organizes the part electrophoretogram of hnRNPF mRNA and FTH1 mRNA amplified production to see the RT-PCR part amplified production electrophoretogram of Fig. 5 hnRNPF mRNA and FTH1 mRNA.A:hnRNPF mRNA part amplified production electrophoretogram, B:FTH1 mRNA part amplified production electrophoretogram; M:DL2000 mark, 1,2,3,4 are breast cancer focus tissue, and 5,6 is mammary gland cancer beside organism, and 7,8 are the mammary gland benign tumor tissue, 9 negative contrasts: replace substrate with deionized water.
HnRNPF mRNA and FTH1 mRNA are in breast cancer focus tissue, cancer beside organism, and mammary gland benign tumor organizes positive expression to see table 12.
Table 12 hnRNPF mRNA and the FTH1 mRNA positive expression [% (routine number)] in different grouping
| Divide into groups | hnRNPF mRNA | FTH1 mRNA |
| Mammary gland benign tumor tissue (32 example) | 18.8% (6)? | 25.0% (8)? |
| Mammary gland cancer beside organism (40 example) | 35.0% (14)? | 27.5% (11)? |
| Breast cancer tissue's (40 example) | 65.0% (26) | 60.0% (24) |
HnRNPF mRNA and FTH1 mRNA are in breast cancer focus tissue, cancer beside organism, and mammary gland benign tumor is organized the positive rate difference analysis: breast cancer group hnRNPF mRNA and FTH1 mRNA positive rate all be significantly higher than expression in cancer beside organism and the mammary gland benign tumor tissue (
P＜0.05), specifically relatively sees Fig. 6
HnRNPF mRNA and the expression of FTH1 mRNA in different tissues
Serum hnRNPF antibody, FTH1 detection of antibodies
HnRNPF antibody, FTH1 antibody are seen table 13 in patient with breast cancer, other cancer patients and normal control expression of serum.
Table 13 hnRNPF antibody and the FTH1 antibody positive expression [% (routine number)] in different grouping
| Divide into groups | HnRNPF antibody | FTH1 antibody |
| Normal control group (54 example) | 22.2% (12)? | 18.5% (10)? |
| Other cancer patients (40 example) | 40.0% (16)? | 27.5% (11)? |
| Patient with breast cancer's (40 example) | 62.5% (25) | 50.0% (20) |
HnRNPF antibody, FTH1 antibody is in patient with breast cancer, other cancer patients and normal control seroprevalence diversity ratio: breast cancer group serum hnRNPF antibody and FTH1 antibody positive rate be significantly higher than other cancer groups and normal control group (
P＜0.05), specifically relatively sees Fig. 7
Two indexes is serum and the variance analysis of healthy women expression of serum before patient with breast cancer's art
The correlativity that the breast cancer candidate markers is expressed in cancer kitchen range tissue and serum
The cancer kitchen range organizes the expression of hnRNPF mRNA, FTH1 mRNA and serum hnRNPF antibody, FTH1 antibody to adopt the Pearson correlation analysis, the expression of two indexes have obvious correlativity (
P=0.01), related coefficient (Pearson index) is 0.643.
The The positive expression rate statistical data of breast cancer specifically expressing index in same sample seen appendix.The consistance that candidate markers is expressed in breast cancer kitchen range tissue and the serum is seen table 14.
The consistance [% (routine number)] that table 14 hnRNPF and FTH1 RT-PCR and ELISA detect
| Index | ?RT-PCR(＋ )ELISA（＋） | RT-PCR(－ )ELISA（－） | Consistance |
| hnRNPF | 60.0%(36) | 30.0% (12) | 90.0% (36) |
| FTH1 | 45.0%(18) | 37.5%(15) | 82.5% (33) |
The cancer kitchen range is organized hnRNPF mRNA, FTH1 mRNA and serum hnRNPF antibody, the variance analysis of FTH1 antibody positive rate: the cancer kitchen range organize hnRNPF mRNA, FTH1 mRNA positive rate and serum hnRNPF antibody, FTH1 antibody positive rate no significant difference (
P＞0.05) (Fig. 8
The expression ratio of two indexes in serum and cancer kitchen range tissue).
(7) value analysis of serum hnRNPF antibody, FTH1 antibody diagnosis breast cancer
The clinical examination result is adopted in the expression of change of serum C A15-3 in the experiment.HnRNPF antibody, FTH antibody and three kinds of indexs of CA15-3 TG-AUC (AUC) independent or joint-detection is seen Fig. 9 Figure 10: FTH (AUC) minimum, and the AUC of hnRNPF, FTH antibody and CA15-3 joint-detection is maximum; Obtain optimal clinical diagnosis critical point simultaneously: hnRNPF antibody is 0.216, and the susceptibility of diagnosis this moment is 68.5%, specificity is 70.3%; FTH1 antibody is 0.253, and the susceptibility of this moment diagnosis is 66.6%, specificity is 76.6%; CA15-3 is 29.6 u/ml, and the susceptibility of this moment diagnosis is 58.2%, specificity is 92.0%.(AUC and 95%CI) and three joint-detection there was no significant differences when hnRNPF antibody and CA15-3 joint-detection (
P＞0.05), and greater than arbitrary individual event detect (AUC and 95%CI) (
P＜0.05).Individual event detects diagnostic index (susceptibility+specificity) maximum of CA15-3, diagnostic index (susceptibility+specificity) and three joint-detection there was no significant differences when hnRNPF antibody and CA15-3 joint-detection (
P＞0.05), significantly greater than individual event detect CA15-3 diagnostic index (
P＜0.05).
(8) serum hnRNPF antibody, FTH1 antibody are worth the early diagnosis of breast cancer
Early-stage breast cancer, non-early-stage breast cancer and normal control group serum hnRNPF antibody, FTH1 antibody positive rate are seen table 15.
Table 15 hnRNPF, FTH1 and hnRNPF merge FTH1 positive rate [% (routine number)] in different grouping
| Group | HnRNPF antibody | FTH antibody |
| Normal control group (27 example) | 22.2% (6)? | 18.5% (5)? |
| Early-stage breast cancer (33 example) | 60.6% (20)? | 54.5% (18)? |
| Non-early-stage breast cancer (187 example) | 67.9% (127) | 58.3% (109) |
Serum hnRNPF antibody, FTH1 antibody is patient with breast cancer, non-early-stage breast cancer patient and the variance analysis of normal control group positive rate in early days: hnRNPF antibody, FTH1 antibody all have between patient with breast cancer's serum and the normal control group patient in early days significant difference (
P＜0.05), in early days between patient with breast cancer's serum and the non-early-stage breast cancer patient all no significant difference (
P＞0.05 (sees Figure 11
The value of two indexes early diagnosis breast cancer).
(9) serum hnRNPF antibody, FTH1 antibody and breast cancer relation by stages
Each clinical stages patient with breast cancer serum hnRNPF antibody, FTH1 antibody positive rate are seen table 16.
Table 16 hnRNPF, FTH1 and hnRNPF merge FTH1 positive rate [% (routine number)] in different clinical stagess
| Clinical stages |
HnRNPF | FTH1 antibody | |
| 0～I phase (58 example) | 48.3% (28)? | 44.8% (26)? | |
| The II phase (73 example) | 71.2% (52)? | 63.0% (46) | |
| The III phase (56 example) | 67.9% (38) | 66.1% (37) | |
| The IV phase (27 example) | 74.1% (20) | 70.4% (19)? |
HnRNPF antibody, FTH1 antibody are in patient with breast cancer's positive rate variance analysis of different clinical stagess: II phase primary breast cancer patients serum hnRNPF antibody, FTH1 antibody all be higher than 0～I phase patient (
P＜0.05), but hnRNPF antibody, FTH1 antibody between II phase, III phase and IV primary breast cancer patient group relatively no significant difference (
P＞0.05) (sees Figure 12).
(10) relation of serum hnRNPF antibody, FTH1 antibody and breast cancer pathology type
Different pathological types patient with breast cancer's serum hnRNPF antibody, FTH1 antibody positive rate are seen table 17.
Table 17 hnRNPF, FTH1 and hnRNPF merge FTH1 positive rate [% (routine number)] in different pathological types
| Histological type | HnRNPF antibody | FTH antibody |
| IDC (189 example) | 73.0% (138) | ?64.6% (122)? |
| ILC (26 example) | 61.5% (16) | 53.8% (14) |
| DCIS (17 example) | ?52.3% (9) | 47.1% (8) |
Serum hnRNPF antibody, FTH1 antibody are in the variance analysis of different pathological types patient with breast cancer positive rate: IDC patients serum hnRNPF antibody, all a little higher than ILC of FTH1 antibody and LCIS, but between each histological type all no significant difference (
P＞0.05) (sees Figure 13).
(11) relation of serum hnRNPF antibody, FTH1 antibody and Metastasis in Breast Cancer
Patient with breast cancer's serum hnRNPF antibody, FTH1 antibody positive rate are seen table 17 after preceding patient with breast cancer of lymphatic metastasis and the lymphatic metastasis.
Table 17 hnRNPF, FTH1 and hnRNPF merge FTH1 positive rate [% (routine number)] in each group
| Whether shift | HnRNPF antibody | FTH antibody |
| Do not see transfer (78 example) | 53.8% (42) | 51.3% (40)? |
| Shift (116 example) | 75.9% (88) | 65.5% (76) |
Serum hnRNPF antibody, FTH1 antibody are in lymphatic metastasis group and not metastasis group patient with breast cancer positive rate variance analysis: lymphatic metastasis group patient with breast cancer serum serum hnRNPF antibody, FTH1 antibody all are higher than not metastasis group patients serum's expression, difference have statistical significance (
P＜0.05) (sees Figure 14).
7, conclusion: hnRNPF and FTH1 be equal high expressed in breast cancer focus tissue and in the serum, and both have very strong relevance.
Breast cancer focus group, mammary gland cancer beside organism, mammary gland benign tumor tissue; HnRNPF mRNA The positive expression rate is respectively 65.0% (26/40), 35.0% (14/40), 18.8% (6/32); Its expression at breast cancer focus tissue is higher than cancer beside organism and mammary gland benign tumor tissue respectively, and difference all has statistical significance (P＜0.05).Patient with breast cancer's serum hnRNPF antibody, FTH1 antibody, positive rate is respectively 62.5% (25/40), 50.0% (20/40).It is 92.5% (37/40) that lesion tissue hnRNPF mRNA expresses the consistance of expressing with serum antibody; It is 80.0% (32/40) that lesion tissue FTH1 mRNA expresses the consistance of expressing with serum antibody.Through the ROC tracing analysis, hnRNPF antibody optimal clinical diagnosis critical point (Cut-Off value) is 0.216, and the susceptibility of its diagnosis at this moment is respectively 68.5%, specificity is respectively 77.7%, accuracy is respectively 70.0%.
The diagnosing tumor mark is the specificity substance that tumour cell itself exists or secretes.Because therefore the tear-away entering body fluid circulation of cancer cell also can detect tumor markers through detecting body fluid such as blood, cerebrospinal fluid, pleural effusion, seroperitoneum.Two indexes correlation analysis P=0.01 in this research, related coefficient (Pearson index) is 0.643, prompting cancer kitchen range organizes positive rate and the serum hnRNPF antibody of hnRNPF mRNA, FTH1 mRNA, the expression of FTH1 antibody that stronger correlativity is arranged.The cancer kitchen range is organized positive rate and serum hnRNPF antibody, the FTH1 antibody positive rate there was no significant difference (P＞0.05) of hnRNPF mRNA, FTH1 mRNA; Two indexes is expressed concordance rate and is reached 90% in cancer kitchen range tissue and serum, biopsy pathology diagnosis of prompting cancerous tissue and serology detect the diagnosis that all helps breast cancer.The most definite diagnostic method of cancer is that pathology is made a definite diagnosis after drawing materials through tissue; But because tissue is drawn materials is that the inspection of wound property is arranged, and can not accomplish to be used for conventional health check-up examination and come cancer diagnosis, so this method has certain limitation; And the serology detection has simple to operate; Antibody can the long period in the serum preservation, patient are easy to advantages such as acceptance, have and are equal to or during suitable diagnostic value, serology is detected as the method into first-selection when detecting cancer kitchen range tissue and blood serum tumor markers.This result of study is pointed out detection serum hnRNPF antibody, FTH1 antibody when examination, tentative diagnosis breast cancer, can replace tissue to draw materials and is detected hnRNPF mRNA, FTH1 mRNA; Make and before not doing pathologic sampling, well breast cancer is made tentative diagnosis or antidiastole, can be in time, better service is in clinical and patient.
Three, the serum antibody of hnRNPF and FTH1 is as the clinical verification of early diagnosing mammary cancer mark
1, specimen collection
As aforementioned, patient with breast cancer's serum specimen is totally 273 examples, divides into groups according to clinical stages; Getting normal women of 54 examples and 40 examples simultaneously suffers from other tumours (wherein lung cancer 6 examples, liver cancer 6 examples, cancer of the stomach 6 examples, colorectal cancer 10 examples, cervical carcinoma 4 examples, carcinoma of endometrium 4 examples, oophoroma 4 examples) female patient serum sample and preserves subsequent use as 80 ℃ of ultra low temperature freezers of control group ， –.
2, the preparation of hnRNPF elisa plate and detection
Utilize the phagocytosis body fluid of expressing the breast cancer diagnosis mark, or the commercialization purchase, preparation hnRNPF elisa plate, detect at patient with breast cancer, other cancer patients and normal control group serum:
(1) dosing: use breast cancer T7 bacteriophage specifically expressing hnRNPF antigen and process T7 phagocytosis body fluid, perhaps directly the hnRNPF antigen liquid is bought in commercialization, and perhaps directly commercialization is bought hnRNPF antigen powder and processed the hnRNPF antigen liquid;
(2) preparation of hnRNPF elisa plate: with T7 phagocytosis body fluid or hnRNPF antigen liquid, encapsulate in ELISA Plate, 4 ℃ are spent the night; T7 phagocytosis body fluid or hnRNPF antigen liquid can dilute usefulness; As dilute more than the 1:100, to reduce cost, the present embodiment dilute strength is l:1000; The hole count of ELISA Plate is unrestricted, and present embodiment is 96 holes; After washing, with 5%BSA/PBS room temperature sealing 2 hours; The aforesaid liquid amount is different because of the purity of material, and specifically the amount of choosing can be decided with reference to the existing general knowledge of this area;
(3) specimen collection and processing: take out peripheral blood to be measured, separate test serum, present embodiment extracts the 5 pipe mixed serum of breast cancer and each 100 μ L of control serum;
(4) detection of hnRNPF elisa plate: the test serum incubated at room of adding 1:100 dilution in every hole of ELISA Plate 1 hour; The washing back adds the goat anti human IgG of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; Test serum amount and goat anti human IgG amount are unrestricted, and roughly the same with both is preferable, and present embodiment is 100 μ l; The washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃;
(5) read plate: 450 nm wavelength read the OD value under ELIASA, and triplicate was all wanted in every hole when the ELISA method detected;
(6) according to the feminine gender of OD value, positive judged result: with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole, judge ratio >=2.0 according to the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control; Be judged as the positive, ratio＜2.0 are judged as feminine gender; Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
3, the preparation of FTH1 elisa plate and detection
Utilize the phagocytosis body fluid of expressing the breast cancer diagnosis mark, or the commercialization purchase, preparation FTH1 elisa plate, detect at patient with breast cancer, other cancer patients and normal control group serum:
(1) dosing: use breast cancer T7 bacteriophage specifically expressing FTH1 antigen and process T7 phagocytosis body fluid, perhaps directly the FTH1 antigen liquid is bought in commercialization, and perhaps directly commercialization is bought FTH1 antigen powder and processed the FTH1 antigen liquid;
(2) preparation of FTH1 elisa plate: with T7 phagocytosis body fluid or FTH1 antigen liquid, encapsulate in ELISA Plate, 4 ℃ are spent the night; T7 phagocytosis body fluid or FTH1 antigen liquid can dilute usefulness; As dilute more than the 1:100, to reduce cost, the present embodiment dilute strength is l:1000; The hole count of ELISA Plate is unrestricted, and present embodiment is 96 holes; After washing, 5%BSA/PBS room temperature sealing 2 hours; The aforesaid liquid amount is different because of the purity of material, and specifically the amount of choosing can be decided with reference to the existing general knowledge of this area;
(3) specimen collection and processing: take out peripheral blood to be measured, separate test serum, present embodiment extracts the 5 pipe mixed serum of breast cancer and each 100 μ L of control serum;
(4) detection of FTH1 elisa plate: the test serum incubated at room of adding 1:100 dilution in every hole of ELISA Plate 1 hour; The washing back adds the goat anti human IgG of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; Test serum amount and goat anti human IgG amount are unrestricted, and roughly the same with both is preferable, and present embodiment is 100 μ l; The washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃;
(5) read plate: 450 nm wavelength read the OD value under ELIASA, and triplicate was all wanted in every hole when the ELISA method detected;
(6) according to the feminine gender of OD value, positive judged result: with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control, with the well that do not encapsulate bacteriophage as blank; After the blank well zeroing, judge ratio >=2.0 according to the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control; Be judged as the positive, ratio＜2.0 are judged as feminine gender; Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
4, the preparation and the detection of hnRNPF elisa plate associating FTH1 elisa plate
As previously mentioned; Diagnostic reagent can be a diagnosis marker with hnRNPF or FTH1 separately; The corresponding antibody of hnRNPF or FTH1 in the test serum that detect to separate can hnRNPF associating FTH1 be diagnosis marker also, detects the corresponding antibody of hnRNPF and FTH1 in the test serum that separates.When the preparation of hnRNPF elisa plate associating FTH1 elisa plate when detecting, its preparation method is with above-mentioned 2 and 3, on associated detecting method in the 6th goes on foot the judged result step variant, all the other are identical.Judged result step in the 6th step of joint-detection: after the blank well zeroing; Ratio according to the average OD value of FTH1 elisa plate test serum OD value/negative control; In conjunction with the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control, judge.The determination methods here has multiple, and the medical science standards different according to this area can have different determination methods.Through clinical; Present embodiment is adopted following preferred embodiment: be specially the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control or the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control; Arbitrary ratio >=2.0 promptly are judged as the positive.Thereby obviously improve the breast cancer diagnosis effect.
5, the clinical value of hnRNPF, FTH1 diagnosing mammary cancer
Among the 273 routine patient with breast cancers; Clear and definite clear and definite 194 examples that whether shift of 232 examples, axillary lymphatic metastasis of 214 examples, histological type that clinical stages is clear and definite; Analyze the visible accompanying drawing 15 of relation, Figure 16 and the table 18 of its serum hnRNPF antibody and FTH1 detection of antibodies result and pathological staging, histological type and axillary lymphatic metastasis: after the breast cancer II phase than 0～I phase, hnRNPF antibody, FTH1 antibody expression positive rate significantly raise (P＜0.05); Shift the patient and do not shift the patient, hnRNPF antibody, FTH1 antibody expression positive rate significantly raise (P＜0.05).
Table 18 hnRNPF antibody, FTH1 antibody positive rate in each group
| Divide into groups | HnRNPF antibody positive rate (%) | FTH1 antibody positive rate (%) |
| Clinical stages | ? | ? |
| 0～I phase | 48.3% (28/58) | 44.8% (26/58)? |
| The II phase | 71.2% (52/73) ▲ | 63.0% (46/73) ▲ |
| The III phase | 67.9% (38/56) ▲ | 66.1% (37/56) ▲ |
| The IV phase | 74.1% (20/27) ▲ | 70.4% (19/27) ▲ ? |
| Histological type | ? | ? |
| IDC | 73.0% (138/189) | ?64.6% (122/189)? |
| ILC | 61.5% (16/26) | 53.8% (14/26) |
| DCIS | 52.3% (9/17) | 47.1% (8/17) |
| Axillary lymphatic metastasis | ? | ? |
| Do not see transfer | 53.8% (42/78) | 51.3% (40/78)? |
| Shift | 75.9% (88/116) ★ | 65.5% (76/116) ★ |
Annotate:
▲With 0～I phase group relatively, difference have statistical significance (
P＜0.05),
★With see metastasis group relatively, difference have statistical significance (
P＜0.05).
Other concrete hnRNPF, FTH1 are in the clinical value of diagnosing mammary cancer, can repeat no more referring to aforementioned two, hnRNPF, the FTH1 specificity verification portion as markers for breast cancer here.
Above embodiment is not the restriction to this case technical scheme only as further specifying of the present invention, and all equivalent variations of doing according to the key problem in technology of this case all fall into the protection domain of this case.
Claims (6)
1. early-stage breast cancer serodiagnosis reagent; It is characterized in that: said diagnostic reagent is with FTH1; Perhaps FTH1 associating hnRNPF is a diagnosis marker; Can detect FTH1 antibody in the test serum of separation, perhaps the corresponding antibody of FTH1 and hnRNPF is used for early diagnosing mammary cancer or postoperative curative effect monitoring.
2. early-stage breast cancer serodiagnosis reagent; It is characterized in that: comprise the FTH1 elisa plate; The FTH1 antigen embedding ELISA Plate that this FTH1 elisa plate is buied by the FTH1 antigen of using breast cancer T7 bacteriophage specifically expressing or commercialization earlier; 4 ℃ are spent the night, and the 5%BSA/PBS room temperature sealing of washing back formed in 2 hours; Also comprise goat anti human IgG, developer A, B and the stop buffer that can cooperate the HRP coupling that the FTH1 elisa plate detects test serum; The FTH1 elisa plate can read the OD value in 450 nm wavelength by ELIASA, with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole, judge ratio >=2.0 according to the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control; Be judged as the positive, ratio＜2.0 are judged as feminine gender; Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
3. a kind of early-stage breast cancer serodiagnosis reagent as claimed in claim 2; It is characterized in that: also comprise the hnRNPF elisa plate; The hnRNPF antigen embedding ELISA Plate that this hnRNPF elisa plate is buied by the hnRNPF antigen of using breast cancer T7 bacteriophage specifically expressing or commercialization earlier; 4 ℃ are spent the night, and the 5%BSA/PBS room temperature sealing of washing back formed in 2 hours; Also comprise goat anti human IgG, developer A, B and the stop buffer that can cooperate the HRP coupling that the hnRNPF elisa plate detects test serum; The hnRNPF elisa plate can read the OD value in 450 nm wavelength by ELIASA, with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole; Ratio according to the average OD value of hnRNPF elisa plate test serum OD value/negative control; Ratio in conjunction with the average OD value of FTH1 elisa plate test serum OD value/negative control; Judge that negative control OD value is lower than 0.05 and does 0.05 calculating, be higher than 0.05 by actual OD value calculating.
4. method of carrying out early diagnosing mammary cancer or postoperative curative effect monitoring is characterized in that: may further comprise the steps:
(1) dosing: use breast cancer T7 bacteriophage specifically expressing FTH1 antigen and process T7 phagocytosis body fluid, perhaps directly the FTH1 antigen liquid is bought in commercialization, and perhaps directly commercialization is bought FTH1 antigen powder and processed the FTH1 antigen liquid;
(2) preparation of FTH1 elisa plate: T7 phagocytosis body fluid or FTH1 antigen liquid are encapsulated in ELISA Plate, and 4 ℃ are spent the night; After washing, 5%BSA/PBS room temperature sealing 2 hours;
(3) specimen collection and processing: take out peripheral blood to be measured, separate test serum;
(4) detection of FTH1 elisa plate: the test serum incubated at room of adding 1:100 dilution in every hole of ELISA Plate 1 hour; The washing back adds the goat anti human IgG of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; The washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃;
(5) read plate: 450 nm wavelength read the OD value under ELIASA;
(6) according to the feminine gender of OD value, positive judged result: with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole, judge ratio >=2.0 according to the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control; Be judged as the positive, ratio＜2.0 are judged as feminine gender; Negative control OD value is lower than 0.05 and does 0.05 calculating, is higher than 0.05 by actual OD value calculating.
5. a kind of method of carrying out early diagnosing mammary cancer or postoperative curative effect monitoring as claimed in claim 4 is characterized in that: further comprising the steps of:
(1) dosing: use breast cancer T7 bacteriophage specifically expressing hnRNPF antigen and process T7 phagocytosis body fluid, perhaps directly the hnRNPF antigen liquid is bought in commercialization, and perhaps directly commercialization is bought hnRNPF antigen powder and processed the hnRNPF antigen liquid;
(2) preparation of hnRNPF elisa plate: T7 phagocytosis body fluid or hnRNPF antigen liquid are encapsulated in ELISA Plate, and 4 ℃ are spent the night; After washing, 5%BSA/PBS room temperature sealing 2 hours;
(3) specimen collection and processing: take out peripheral blood to be measured, separate test serum;
(4) detection of hnRNPF elisa plate: the test serum incubated at room of adding 1:100 dilution in every hole of ELISA Plate 1 hour; The washing back adds the goat anti human IgG of the HRP coupling of 1:10 000 dilution, and room temperature was placed 1 hour; The washing back adds each 1 of developer A, B, hatches 15 minutes, and adds stop buffer immediately for 37 ℃;
(5) read plate: 450 nm wavelength read the OD value under ELIASA;
(6) according to the feminine gender of OD value, positive judged result: with elder generation after bacteriophage, mouse-anti bacteriophage one are anti-and the sheep anti mouse HRP two anti-wells that encapsulate as negative control hole, with the well that do not encapsulate bacteriophage as the blank hole; After the zeroing of blank hole; Ratio according to the average OD value of hnRNPF elisa plate test serum OD value/negative control; Ratio in conjunction with the average OD value of FTH1 elisa plate test serum OD value/negative control; Judge that negative control OD value is lower than 0.05 and does 0.05 calculating, be higher than 0.05 by actual OD value calculating.
6. a kind of method of carrying out early diagnosing mammary cancer or postoperative curative effect monitoring as claimed in claim 5; It is characterized in that: in said (6) step according to the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control; Ratio in conjunction with the average OD value of FTH1 elisa plate test serum OD value/negative control is judged; Be meant the ratio of the average OD value of hnRNPF elisa plate test serum OD value/negative control or the ratio of the average OD value of FTH1 elisa plate test serum OD value/negative control; Arbitrary ratio >=2.0 promptly are judged as the positive.
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