CN105277718B

CN105277718B - For the product of the examination of malignant tumour correlation and assessment, application and method

Info

Publication number: CN105277718B
Application number: CN201510632081.1A
Authority: CN
Inventors: 任士芳; 顾建新; 张泽建; 周蕾; 孙琳琳
Original assignee: Shanghai Zhixian Biological Technology Co Ltd
Current assignee: Shanghai Zhixian Biological Technology Co Ltd
Priority date: 2015-09-29
Filing date: 2015-09-29
Publication date: 2018-03-20
Anticipated expiration: 2035-09-29
Also published as: WO2017054686A1; CN105277718A

Abstract

The invention provides for the product of the examination of malignant tumour correlation and assessment, application and method, and in particular to a kind of product, application and method for carrying out examination, early diagnosis, prognosis evaluation, risk assessment, state of illness monitoring and/or curative effect evaluation to malignant tumour by detecting immunoglobulin G surface double antenna complexity N sugar-chain ends galactolipin change in blood.

Description

For the product of the examination of malignant tumour correlation and assessment, application and method

Technical field

The invention belongs to biotechnology and medical domain.Specifically, exempted from the present invention relates to one kind by detecting in blood The double antenna complexity N sugar-chain ends galactolipin change of epidemic disease Lysozyme surface carries out examination, early diagnosis, prognosis to malignant tumour Assessment, risk assessment, the product and method of state of illness monitoring and/or curative effect evaluation.

Background technology

Tumour is one of disease of world today's serious threat human health, and it has long-time, multistage feature.When When patient has the subjective symptoms to go to see a doctor, tumour has often had developed to middle and advanced stage, is brought to clinical treatment very big tired It is difficult.And the death rate of Advanced cancers is very high, five year survival rate often only has 20%.If in early detection tumour and it can monitor Its development and change, treatment, the five year survival rate of tumour then can reach 80% in time.At present, medical field generally believes secondary prevention (early to find, early diagnose, early treatment) is one of key link for reducing cancer mortality.It is noted that fallen ill from the age Rate sees that the incidence of disease rises obvious after 45 years old.Therefore, examination was carried out for malignant tumour since 40 years old, there is very big society Can meaning.

At present, the examination means of malignant tumour mainly include imageological examination and blood serum tumor markers examination.

Imageological examination including CT, nuclear magnetic resonance, endoscope etc. is on the one hand because its is costly, operation is professional The problems such as cause its be difficult to large area as examination means popularize, on the other hand at present these conventional imageological examination means be It is identified for tissue morphology, can only often detects a certain size existing Advanced cancers.

In view of the convenience of Blood specimen collection, serology lesion detection means are more suitable for extensive tumor screening.So And some existing blood serum tumor markers, including alpha-fetoprotein (AFP), cancer antigen 125 (CA125), carcinomebryonic antigen at present (CEA), cancer antigen 15-3 (CA15-3) and prostate specific antigen (PSA) etc., their Sensitivity and Specificity be not universal high. Even AFP the most sensitive, the positive rate that it is diagnosed is also less than 70%.On the other hand, these tumor markerses are substantially It is difficult Comprehensive consideration during for detecting for a certain or certain a kind of tumour.

In addition, the prognosis of malignant tumour and people at highest risk occur the assessment of malignant tumour risk, conditions of patients monitoring and/ Or generation of the curative effect evaluation for clinic control malignant tumour, recurrence, treatment etc. also have great importance.

In this context, a kind of broad covered area, sensitiveness height, specific good, the hematology side of simple operation are found out Early screening, prognosis evaluation, prediction (i.e. risk assessment), state of illness monitoring and/or the curative effect evaluation that method is used for malignant tumour seem It is very urgent.

Sugar chain almost take part in all life processes, the change of sugar chain structure and disease as a kind of biological information molecule Generation, the development of the especially major disease such as tumour, autoimmune disease, infection are closely related.Swollen without obvious sign Knurl early period of origination, glycosylation occurred for some glycoprotein abnormal on cell membrane, and is discharged into blood, turns into tumour early detection Important clue.Kinds of tumors serodiagnosis biomarker such as CA19-9 (tumor in digestive tract), AFP (liver cancer), CA125 (ovum Nest cancer) etc. be tumour occur after body secretes sugar chain or glycoprotein product.

In a word, the exploitation deeply for new haematological tumours detection method based on sugared area research provides opportunity.

The content of the invention

Inventor has found that the terminal galactosylation degree of double antenna complexity N sugar chains on human serum IgG (is specially N sugar Chain end removes galactolipin (Gal₀) and galactolipin sugar chain (Gal₁And Gal₂) abundance ratio) with the generation of kinds of tumors, good pernicious And prognosis has universal correlation.Based on this, the invention provides new malignant tumour examination, prognosis evaluation, risk assessment, The method and product of state of illness monitoring and/or curative effect evaluation.

In the first aspect of the present invention, there is provided one kind is commented for the examination of object malignant tumour, early diagnosis, prognosis Estimate, risk assessment, the product of state of illness monitoring and/or curative effect evaluation, the product can be such as kit, equipment, operable system System and/or combinations thereof, the product include：

(A) it is used for reagent, the instrument for determining the terminal galactosylation degree of blood IgG surfaces double antenna complexity N sugar chains Device and/or system, it is used to determine the double antenna complexity N- sugar chain abundance Gal that IgG face extremities connect without galactolipin₀, end End is connected with the double antenna complexity N- sugar chain abundance Gal of 1 galactolipin₁The double antenna complexity of 2 galactolipins is connected with end N- sugar chain abundance Gal₂；

For example, for reagent, instrument and/or the system of the one or more methods being selected from the group：Ground substance assistant laser solution Analyse time-of-flight mass spectrometry (TOFMS) MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, electrospray ionization mass spectrum ESI-MS；Liquid chromatography；Liquid Phase chromatograph-mass spectrometer coupling method；Carbohydrate chip technology；Nuclear magnetic resonance nmr, preferred substrate Assisted Laser Desorption flight time mass spectrum MALDI methods；

(B) it is used to calculate Gal₀With Gal₁And Gal₂Abundance ratio Gal than module and/or processor；

(C) optionally, for according to Gal than judge object whether suffer from malignant tumour, to object progress prognosis evaluation and/ Or suffer from the module and/or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation.

In certain embodiments of the present invention, the blood is selected from：Serum, blood plasma and whole blood, preferably serum.

In certain embodiments of the present invention, the product also includes the one or more being selected from the group：

A) it is used for the reagent and/or instrument for gathering and/or dealing with objects blood sample；

B) it is used for separation and/or the reagent and/or instrument of purified blood serum and/or blood plasma；

C) it is used for the reagent and/or instrument of separation and/or purified blood serum/blood plasma IgG；

D) it is used for the reagent and/or instrument for separating, purifying and/or being enriched with serum IgG surface N- sugar chains,

For example, what is be selected from the group isolates and purifies the reagent and/or instrument of N sugar chain methods：Porous graphite carbon PGC solid phases extract Follow the example of (preferably using acetonitrile:Water:Trifluoracetic acid=80:19.9:0.1 activation PGC, using water:Trifluoracetic acid=99.9:0.1 is flat Weigh PGC, using acetonitrile:Water:Trifluoracetic acid=25:74.95:0.05 mixed solvent elution sugar chain), polysaccharide purification post, aggegation Plain affinity method (such as continuous agglutinin affinity chromatography SLAC), Capillary Electrophoresis, high performance liquid chromatography；

E) the double antenna complexity N sugar-chain ends for being used to store and/or deal with objects serum remove galactolipin and galactolipin sugar Database, module and/or processor of the chain abundance ratio as IgG surfaces glycosyl galactose quantitative change index；

F) it is used for the module and/or processor that judgment threshold is provided；

G) it is used for the Gal ratios of object and compares to judge whether object suffers from malignant tumour, object is carried out in advance Assess and/or suffer from afterwards the module and/or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation；

H) it is used to provide diagnosis and/or testing result and/or the module and/or processor of report；

J) specification or guide for use, wherein describing following one or more applications and judgment mode：

(i) it is used to tumor screening detect：When Gal ratios reach at or above threshold value set in advance, then judge that the subject suffers from There is malignant tumour or with the risk for suffering from malignant tumour；

(ii) it is used to good dislike judge：When Gal ratios reach at or above threshold value set in advance, then judge the subject with disliking Property tumour or with suffering from the risk of malignant tumour；

(iii) it is used for Prognosis scoveillance：When Gal ratios reach at or above threshold value set in advance, then object malignant tumour is prompted Recurrence or the excessive risk with recurrence；

(iv) it is expected for groups of objects (such as high risk group) malignant tumour：When Gal ratios reach at or above advance set Fixed threshold value, then prompt object that the possibility of malignant tumour occurs and be higher than other objects；

(v) it is used for cancer patient's state of illness monitoring：When Gal is more advance than being reached at or above higher than the Gal ratios obtained by previous test The scope (such as 5%~50% or the arbitrfary point in the range of this or subrange) of setting, then showing the state of an illness of the cancer patient has Develop or further deteriorate；

(vi) it is used for curative effect evaluation：Different time points after before the treatment or in therapeutic process determine and calculate Gal ratios, When Gal ratios reach at or above scope set in advance (such as 5%~50% or should higher than the Gal ratios obtained by previous test In the range of arbitrfary point or subrange), then the effect of showing the therapy and/or medicine, is bad；

It is preferred that the threshold value in (i)~(iv) is 0.35~0.6, more preferably 0.5.

In some embodiments, can be based on maximum slope point in ROC curve or maximum sensitivity and the value of specificity To determine optimal threshold, and preferable threshold range can be optimal threshold ± 1~15% (including any number point in the range of this Or subrange) scope.

In some embodiments, the control is selected from：Obtained from Healthy People or the sample or default of non-malignant patient Threshold value.

In certain embodiments of the present invention, the Gal₀With Gal₁And Gal₂Abundance ratio calculating using being selected from The formula of the following group is carried out：

Gal ratios=Gal₀/(Gal₁+Gal₂)；

Gal ratios=Gal₀/(Gal₁+2Gal₂)；Or

Other variations such as its inverse, logarithm.

In certain embodiments of the present invention, the malignant tumour is selected from：Liver cancer, stomach cancer, lung cancer, oophoroma, intestinal cancer, The cancer of the esophagus, cancer of pancreas, cholangiocarcinoma, kidney, breast cancer, prostate cancer, nasopharyngeal carcinoma, carcinoma of urinary bladder, carcinoma of endometrium and cervical carcinoma.

In certain embodiments of the present invention, the malignant tumour is in the stage being selected from the group：Precancerous lesion, clinic Preceding cancer phase, early-stage cancer, cancer middle and advanced stage.

In certain embodiments of the present invention, the object is mammal, preferably people, monkey, dog, horse, ox, sheep, pig, Mouse, rabbit etc., more preferably cancer people at highest risk.

In certain embodiments of the present invention, the product for high flux detect, such as simultaneously handle 96, 192nd, 288,384,480,576 or more samples.

In certain embodiments of the present invention, the product is to contain in the detection method of following steps：

(A') terminal galactosylation of IgG surfaces double antenna complexity N sugar chains is horizontal in measure object blood sample, institute Stating terminal galactosylation level includes：The horizontal Gal of double antenna complexity N- sugar chains that IgG face extremities connect without galactolipin₀、 End is connected with the horizontal Gal of double antenna complexity N- sugar chains of 1 galactolipin₁The double antenna that 2 galactolipins are connected with end is complicated The horizontal Gal of type N- sugar chains₂；

(B') Gal is calculated₀With Gal₁And Gal₂Abundance ratio, i.e. Gal ratios；

(C') according to Gal than judging whether object suffers from malignant tumour, carry out prognosis evaluation to object and/or suffers from tumour Risk assessment, the state of an illness is monitored and/or curative effect is assessed.

In certain embodiments of the present invention, methods described also includes the one or more steps being selected from the group：

(a') gather and/or deal with objects blood sample；

(b') separation and/or purified blood serum and/or blood plasma；

(c') separation and/or purified blood serum/blood plasma IgG；

(d') separate, purify and/or be enriched with serum IgG surface N- sugar chains；

(e') the double antenna complexity N sugar-chain ends for storing and/or dealing with objects serum remove galactolipin and galactolipin sugar chain Abundance ratio is as IgG surfaces glycosyl galactose quantitative change index；

(f') judgment threshold is provided；

(g') by the Gal ratios of object and compare with judge object whether suffer from malignant tumour, to object carry out prognosis Assess and/or suffer from tumour, state of illness monitoring and/or curative effect evaluation；

(h') diagnosis and/or testing result and/or report are provided；

(j') other malignant tumour indexs are detected, and by the testing result of other malignant tumour indexs and Gal than examining Result is surveyed to combine.

In certain embodiments of the present invention, the product also includes the reagent for being used for detecting other malignant tumour indexs Box, equipment, system and/or combinations thereof, preferably described index are selected from：The basic condition of patient, such as age, sex, family History；Clinical tumor shows, including topical manifestations such as lump and secondary symptom, pain, pathological discharge thing, skin and mucosa ulcer, General manifestation such as heating, infection, anaemia, become thin, be weak, dyscrasia, inside examining such as digital rectal examination, gynecologial examination；Selectivity physics and chemistry Index for examination, including specific image check, such as nuclear-magnetism, CT, B ultrasound, endoscope, breast molybdenum target, tumor biochemistry index, as AFP, CEA、PSA、SF、TSGF、POA、PROGRP、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、 NSE, SCC, AFU, EBV-VCA etc..

In the second aspect of the present invention, there is provided reagent, instrument, module and/or processor are being prepared for object evil Property tumor screening, early diagnosis, prognosis evaluation, risk assessment, state of illness monitoring and/or curative effect evaluation product in application, institute It can be such as kit, equipment, system and/or combinations thereof to state product, the reagent, instrument, module and/or processor bag Include：

(A) it is used for reagent, the instrument of terminal galactosylation level for determining blood IgG surfaces double antenna complexity N sugar chains Device, module and/or processor, it is used to determine the double antenna complexity N- sugar chains level that IgG face extremities connect without galactolipin Gal₀, end be connected with the horizontal Gal of double antenna complexity N- sugar chains of 1 galactolipin₁The double antenna of 2 galactolipins is connected with end The horizontal Gal of complexity N- sugar chains₂；

For example, for reagent, instrument, module and/or the processor of the one or more methods being selected from the group：Matrix-assisted Laser desorption time-of-flight mass spectrometry (TOFMS) MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, electrospray ionization mass spectrum ESI-MS；Liquid chromatogram Method；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip technology；Nuclear magnetic resonance nmr, preferred substrate Assisted Laser Desorption flight time matter Spectrometry MALDI-MS；

(B) optionally, for calculating Gal₀With Gal₁And Gal₂Abundance ratio Gal than module or processor；

(C) optionally, for according to Gal than judge object whether suffer from malignant tumour, to object progress prognosis evaluation and/ Or suffer from the module or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation.

In certain embodiments of the present invention, the product is as previously described.

In the third aspect of the present invention, there is provided a kind of method screened for medicine and/or treatment method, the side Method includes：

(A ") measure object be administered and/or treatment before, afterwards and/or during different time points blood sample in The terminal galactosylation of IgG surfaces double antenna complexity N sugar chains is horizontal, and the terminal galactosylation level includes：IgG tables The horizontal Gal of double antenna complexity N- sugar chains that face end connects without galactolipin₀, end be connected with 1 galactolipin double antenna it is complicated The horizontal Gal of type N- sugar chains₁The horizontal Gal of double antenna complexity N- sugar chains of 2 galactolipins is connected with end₂；

(B ") calculates Gal₀With Gal₁And Gal₂Abundance ratio, i.e. Gal ratios；

(C ") judges the validity of the medicine and/or treatment method according to Gal than assessing, wherein when Gal ratios are higher than Gal ratios obtained by previous test reach at or above scope set in advance (such as 5%~50% or should in the range of arbitrfary point Or subrange), then the effect of showing the medicine and/or treatment method, is bad.

In other aspects of the invention, there is provided one kind is commented for the examination of object malignant tumour, early diagnosis, prognosis Estimate, risk assessment, the method for state of illness monitoring and/or curative effect evaluation, it is characterised in that methods described employs the product of the present invention Or the application of the present invention.

In some embodiments, methods described includes：

(a') gather and/or deal with objects blood sample；

(b') separation and/or purified blood serum and/or blood plasma；

(c') separation and/or purified blood serum/blood plasma IgG；

(f') judgment threshold is provided；

(h') diagnosis and/or testing result and/or report are provided；

(j') other malignant tumour indexs are detected, and by the testing result of other malignant tumour indexs and Gal than examining Survey result to combine, preferably described index is selected from：The basic condition of patient, such as age, sex, family history；Clinical tumor shows, bag Topical manifestations such as lump and secondary symptom, pain, pathological discharge thing, skin and mucosa ulcer are included, general manifestation is as generated heat, sense Dye, anaemia, become thin, be weak, dyscrasia, inside examining such as digital rectal examination, gynecologial examination；Selectivity physico-chemical examination index, including it is specific Image check, such as nuclear-magnetism, CT, B ultrasound, endoscope, breast molybdenum target, tumor biochemistry index, as AFP, CEA, PSA, SF, TSGF, POA、PROGRP、CA125、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、NSE、SCC、AFU、EBV- VCA etc..

In some embodiments, methods described is used for purposes as described below, and including corresponding steps as described below：

In certain embodiments of the present invention, the product and application are used in the method that comprises the following steps：

Separated with IgG purification columns from ring polymer sample and/or purified blood serum IgG, it is preferred to use with purifying column kit Alkalescent combination cushioning liquid and faintly acid elution cushioning liquid；

The IgG separated with glucosides ferment treatment is to separate IgG N sugar chains, it is preferred to use glycosidase PNGase F；

With the N sugar chains of porous graphite carbon PGC Solid phase extraction separations, it is preferred to use acetonitrile:Water:Trifluoracetic acid=80: 19.9:0.1 activation PGC, using water:Trifluoracetic acid=99.9:0.1 balances PGC, using acetonitrile:Water:Trifluoracetic acid=25: 74.95:0.05 mixed solvent elution sugar chain；

Using Gal in mass spectrometry (such as MALDI TOF MS method) quantitative analysis N- sugar chains₀、 Gal₁And Gal₂It is horizontal；

Show that the examination of object malignant tumour, early diagnosis, prognosis evaluation, risk are commented based on ring polymer IgG surface Gas l ratios Estimate, the result of state of illness monitoring and/or curative effect evaluation.

Those skilled in the art can be combined without departing from this hair to foregoing technical scheme and technical characteristic Bright inventive concept and protection domain.The other side of the present invention is due to this disclosure, to those skilled in the art For be obvious.

Brief description of the drawings

The invention will be further described below in conjunction with the accompanying drawings, wherein it is shown only for illustrate the present invention implementation Scheme, rather than in order to limit to the scope of the present invention.

Fig. 1：All kinds of malignant tumour samples and the comparison for compareing (Healthy People and benign disease)；

A：The serum IgG N- sugar chains of control represent mass spectra (numeral above peak represents its molecular weight)；

B：The serum IgG N- sugar chains of malignant tumour sample represent mass spectra；

Fig. 2A：The ROC curve of comparative sample of all kinds of malignant tumour samples with compareing (Healthy People and benign disease)；

Fig. 2 B：ROC curve of the oophoroma sample compared with compareing (Healthy People and benign disease)；

Fig. 2 C：ROC curve of the lung cancer sample compared with compareing (Healthy People and benign disease)；

Fig. 2 D：ROC curve of the stomach cancer sample compared with compareing (Healthy People and benign disease)；

Fig. 2 E：ROC curve of the liver cancer sample compared with compareing (Healthy People and benign disease)；

Fig. 2 F：Intestinal cancer (including large intestine, small intestine, colorectal cancer) sample is compared with compareing (Healthy People and benign disease) ROC curve；

Fig. 2 G：The ROC curve of all kinds of malignant tumour samples and benign disease comparative sample；

Fig. 2 H：ROC curve of the primary malignant tumor patient sample compared with control (health and benign disease) sample.

Subordinate list explanation

Combining subordinate list further below, the invention will be further described, wherein the shown reality only for the explanation present invention Scheme is applied, rather than in order to limit to the scope of the present invention.

Table 1：Check sample is classified and sample age distribution；

Table 2：Malignant tumour sample is by stages and hierarchical statistics；

Table 3A：Gal is than detecting the statistical discrepancy in malignant tumor patient and check sample；

Table 3B：Staging and various index recall rates compare.

Embodiment

The present invention is based on research of the inventor in above-mentioned field, there is provided one kind is based in mammal (preferably people) blood The change examination malignant tumour of immunoglobulin G (IgG) surface double antenna complexity N sugar-chain end glycosyl galactoses, carry out early stage Diagnosis, prognosis evaluation, risk assessment, the new method of state of illness monitoring and/or curative effect evaluation.With existing tumor serology detection side Method is compared, and method of the invention has that applicability is wide, sensitiveness is high, specificity is good, simple operation characteristic, for the morning of tumour Phase discovery, in time diagnosis and treatment, improvement prognosis, raising survival rate have important meaning.

The purpose of the present invention is to be directed to end of the malignant tumour with compareing double antenna complexity N sugar chains in IgG surfaces in blood Galactosylation degree (is specially that N sugar-chain ends remove galactolipin (Gal₀) and galactolipin sugar chain (Gal₁And Gal₂) abundance ratio Value) difference, by the analysis to the glycosyl galactose degree, distinguish malignant tumor patient and health/benign and compare, realize to swelling Early screening, malignant and benign lesion, prognosis detection, state of illness monitoring and/or the curative effect evaluation of knurl, and thus provide it is a kind of it is highly sensitive, The malignant tumour screening method of high specific.

Inventor is had found according to the Gal of the present invention than the generation to kinds of tumors and good pernicious universal related.With it is existing Classical index is compared, the present invention to the tumor screening of first visit sufferer (liver cancer, stomach cancer, lung cancer, oophoroma, intestinal cancer, the cancer of the esophagus, Cancer of pancreas, cholangiocarcinoma, kidney, breast cancer, prostate cancer, nasopharyngeal carcinoma, cervical carcinoma, carcinoma of urinary bladder, carcinoma of endometrium) and good pernicious mirror Not Zhen Duan in, there is higher sensitivity, specificity and stability；When with existing classical index use in conjunction, hence it is evident that carry The recall rate of high malignant tumour.

Specifically, inventor has carried out following test and has drawn corresponding conclusion with the present invention：

1) 2661 clinical samples are detected, include 924 check samples, 1737 malignant tumour samples, it was demonstrated that Method, mark and the index of the present invention has high specific, sensitivity and an accuracy in malignant tumour examination；

2) method of the invention can with classical index conjunctive use, to improve the recall rate in specific tumors；

3) the patients with gastric cancer follow-up visit monitoring of 78 customary specification operation plan treatments is studied, for the recall rate of recurrent cases For 92.0%；

4) serum sample for 4217 people at highest risk carries out follow-up detection, and Gal is than tumour in feminine gender group and positive group Incidence significant difference.

Result above confirms that Gal ratios can be independently used for the examination of malignant tumour, the early detection of malignant tumour, malignant tumour Prognosis, state of illness monitoring and/or curative effect evaluation, also can with classical index conjunctive use be used for malignant tumour detection.

Relational language defines

All number ranges provided herein be intended to clearly include falling all numerical value between endpoints of ranges and it Between number range.The feature that the feature or embodiment that can be mentioned to the present invention are mentioned is combined.This specification is taken off All features shown can be used in combination with any combinations thing form, each feature disclosed in specification, can provide phase with any The alternative characteristics substitution of same, impartial or similar purpose.Therefore except there is special instruction, disclosed feature is only impartial or similar The general example of feature.

As used herein, " containing ", " having " or " comprising " include "comprising", " mainly by ... form ", " substantially By ... form " and " by ... form "；" mainly by ... form ", " substantially by ... form " and " by ... form " Belong to the subordinate concept of " containing ", " having " or " comprising ".

As used herein, term " Gal ratios (Gal ratio) " refers to object blood IgG surfaces double antenna complexity N sugar chains Terminal galactosylation quantitative change, specially double antenna complexity N sugar-chain ends remove galactolipin (Gal₀) and galactolipin sugar chain (Gal₁And Gal₂) abundance ratio, wherein, Gal₀The double antenna complexity N- sugar chains connected corresponding to end without galactolipin, Gal₁The double antenna complexity N- sugar chains of 1 galactolipin, Gal are connected with corresponding to end₂2 galactolipins are connected with corresponding to end Double antenna complexity N- sugar chains.

As used herein, term " double antenna complexity N- sugar chains " refers to：Connected on the core pentasaccharides shared in N- sugar chains Enter the branched carbohydrate chains of 2 non-pure mannoses, just like antenna-like (referring to looking into the good chief editor of tin,《Biochemistry》People's Health Publisher, 2008 the 7th edition, the 454-455 pages).

Exemplary Gal₀、Gal₁And Gal₂Structure is as follows, wherein, GlcNAc represents N-Acetyl-D-glucosamine, Man Mannose is represented, Gal represents galactolipin, and Fuc represents fucose.

As used herein, term " specificity ", " sensitivity ", " coincidence rate " have with corresponding medical statistics technics There is identical implication." specificity " (specificity) refers to be diagnosed as to be detected as the moon through kit in the case of " non-cancer " The ratio of property." sensitivity " (sensitivity) refers in the case that pathological diagnosis is " cancer ", and sun is detected as through kit The ratio of property." coincidence rate " (accuracy) refers to that true positives sample size and true negative sample size sum account for the ratio of total sample size Whether example, reflection kit testing result and subject suffer from the degree that cancered truth is consistent.

As used herein, term " threshold value ", " cut-off values ", " cutoff value ", " dividing value " and " reference value ", can exchange Use, refer to the standard for judging testing result, i.e. critical value, testing result is considered as the positive higher than threshold value, less than threshold Value is considered as feminine gender.

As used herein, term " clinical Benefit ", refer to and reach stable disease or the situation of alleviation by certain treatment, wherein CR, PR and SD have and the identical implication of corresponding term, i.e. CR in solid tumor the standard of curative effect evaluation (RECIST standards) (Complete response) tumor complete relieving, PR (Partial response) tumor section alleviates and SD (Stable Disease) stable disease.This three index defining as clinical Benefit crowd of CR, PR and SD, on the contrary PD (Progressive disease) is progression of disease.

As used herein, term " Healthy People/object " refers to normal without any of benign and malignant disease, appearance, People/object without any visible disease symptomses.

Terms used herein " malignant tumor patient " refers to be diagnosed as the patient with malignant tumour through histopathology report. The malignant tumour includes but is not limited to：Liver cancer, stomach cancer, lung cancer, oophoroma, intestinal cancer, the cancer of the esophagus, cancer of pancreas, cholangiocarcinoma, kidney Cancer, breast cancer, prostate cancer, nasopharyngeal carcinoma, cervical carcinoma, carcinoma of urinary bladder, carcinoma of endometrium.It is described in some examples of the present invention Malignant tumour is in the stage being selected from the group：Early-stage cancer, middle and terminal cancer etc..

As used herein, term " non-cancer related disorder patients " or " benign disease patient " refer to：Through pathological diagnosis (example As clinical biochemical, image or histopathology report) be diagnosed as be not cancer but with benign relevant disease patient, such as in lung cancer Experiment middle finger, which is diagnosed as, not to be lung cancer but suffers from the patient of the benign relevant diseases of lung such as pneumonia, pulmonary tuberculosis, in liver cancer experiment Referring to be diagnosed as is not liver cancer but suffers from the patient of the benign relevant diseases of liver such as hepatitis, hepatic sclerosis, refers in colorectal cancer experiment Being diagnosed as is not colorectal cancer but suffers from the patient of the benign relevant diseases of Colon and rectum such as inflammation, colorectal polypus, is tried in breast cancer It is not lung, breast cancer cancer but the patient for suffering from the Benign Breast relevant diseases such as mastitis, the proliferation of mammary gland to test middle finger and be diagnosed as.

As used herein, term " non-evil sample " refers to：Healthy People and benign disease patient are (i.e. true through pathological diagnosis Examine not to be cancer but suffering from the patient of benign relevant disease).

As used herein, term " people at highest risk/colony " refers to the crowd/colony for having tumorigenic highly dangerous degree, Espespecially according to the conditional filterings such as age and habits and customs come out the conscious crowd for not suffering from tumour, not using clinical indices as Partitioning standards.Tumorigenic possibility is significantly larger than population in the people at highest risk of tumour.By taking lung cancer as an example, inhale for many years The male senile patient of cigarette is people at highest risk；For another example liver cancer, Hepatitis B Virus Infection are people at highest risk.

As used herein, term " malignant tumour examination " refers to：By particular detection method regularly to healthy population/group Body is checked, the possibility patient of appearance health and people/object in inferior clinical symptom are identified, and is expected that by Further diagnostic program energy early detection patient, reach deformity caused by disease occurs or slowed down for prevention disease through early treatment And death, make patient obtain preferable prognosis and life quality (Fang Jiqian, Liu Qing,《Chinese tumour》, 2002,11 (1)：10- 11)。

As used herein, term " diagnosing early malignant tumor " refers to：A kind of diagnosis and treatment specifically for tumour early stage patient Method, early treatment is found its object is to early, so as to mitigate patient suffering and spirit, financial burden, and striven for early by tumour Phase diagnosis, treatment allow tumor patient early recovery.

Blood (including serum, blood plasma and whole blood) IgG and its separation and purifying

In embodiments of the present invention, can acquisition target blood sample and with conventional method as known in the art point From with preserve whole blood, serum and/or blood plasma.Fresh or frozen blood, serum or blood plasma can be used to carry out the test of the present invention.Institute Blood sample is stated available from various mammalian objects, preferably people, monkey, dog, horse, ox, sheep, pig, mouse, rabbit etc..Art technology Personnel are appreciated that due to the difference on composition, are detected respectively when using the plasma sample and serum sample of subject When, Gal ratios may be slightly different.For purpose that is quantitative and comparing is easy to, serum sample is preferable in the present invention.

The method for separating IgG is known to persons of ordinary skill in the art, and methods described includes but is not limited to：IgG is purified Post, salting out method, organic solvent precipitation method, polyethylene glycol displacement method, liquid chromatography, affinity chromatography are (such as albumin A affinity method, poly- Acid amides composite membrane affinity method), as long as methods described, which is not destroyed on IgG, connects N- sugar chains.It is commercially available IgG separation agents Box, such as purchased from Thermo Fisher Scientific.

IgG purification columns are employed in an embodiment of the invention to separate the IgG in sample, it is preferred to use albumen A purification columns, and using the alkalescent combination cushioning liquid and faintly acid elution cushioning liquid with purifying column kit, it is more preferably described Purification column is high flux purification column, such as can handle the purification column of 96 samples simultaneously.

IgG surfaces N- sugar chains and its separation, purifying and analysis

N sugar chains can be separated from IgG using method as known in the art, included but is not limited to：Enzyme process, for example with sugar Glycosides enzyme, preferably glycosidase PNGase F；Chemical method, for example with glycoprotein hydrazinolysis reagent, such as ADM0155A hydrazinolysis kits.

After separating N sugar chains from IgG, N sugar chains can be separated and/or purified using method as known in the art, Methods described includes but is not limited to：Porous graphite carbon PGC solid phase extractions, polysaccharide purification post, agglutinin affinity method are (as continuously Agglutinin affinity chromatography SLAC), Capillary Electrophoresis, high performance liquid chromatography etc..

In an embodiment of the invention, porous graphite carbon PGC solid phase extractions separation N sugar chains are employed, its In employ acetonitrile:Water:Trifluoracetic acid=80:19.9:0.1 activation PGC, using water:Trifluoracetic acid=99.9:0.1 balance PGC, and use acetonitrile:Water:Trifluoracetic acid=25:74.95:(ratio is volume to 0.05 mixed solvent elution sugar chain Than).

Galactolipin can be gone to the horizontal double antenna complexity N sugar-chain ends in IgG surfaces using method as known in the art (Gal₀), galactolipin sugar chain (Gal₁And Gal₂) abundance be measured, as long as methods described can be to glycosyl galactose water in N- sugar chains It is flat to be quantified.Methods described includes but is not limited to：Mass spectrography, such as MALDI TOF MS method MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, electrospray ionization mass spectrum ES-MS；Liquid chromatography；Liquid Chromatography-Mass Spectrometry； Carbohydrate chip technology；Nuclear magnetic resonance nmr or more mode appoints charge-coupled conjunction.

In the present invention preferably using Matrix assisted laser desorption ionization (MALDI) mass spectrum (MS) technology to IgG surfaces The terminal galactose degree of double antenna complexity N sugar chains carries out high flux quick detection.The technology has the following advantages that：

1) the direct quick detection after free to N- sugar chains is realized, with high performance liquid chromatography (HPLC) and liquid phase color Spectrum-mass spectrum (LC-MS) combination is compared, and avoids prolonged chromatographic separation process；

2) compared with the MALDI mass-spectrometric technique analyzing sugar chains of routine, without the derivatization step of sugar chain, not only simplify point Step is analysed, analysis time is saved, is especially that of avoiding the experimental error that derivatization step is brought；

3) galactolipin is gone to refer to the sugar chain abundance ratio containing galactolipin as galactolipin quantitative change using complexity N sugar-chain ends Mark, can avoid deviation caused by the operating process such as the pretreatment of parallel sample, reduce when parallel samples enters Mass Spectrometer Method Error, it is ensured that the high reappearance and accuracy of analysis.In a word, above-mentioned characteristic makes the index possess quickly, flux, accurately special Point, condition is provided as a kind of potential clinical diagnosis index for it.

MALDI TOF MS method MALDI-MS is employed in an embodiment of the invention, It is preferred that mass calibration standard items used in the test are TOFmix, matrix used is DHB DHB and/or used Homogenization reagent is ethanol.

Data analysing method

In the present invention, galactolipin is removed into Gal ratios, the i.e. end of object blood IgG surfaces double antenna complexity N sugar chains (Gal₀) and galactolipin sugar chain (Gal₁And Gal₂) abundance ratio, as malignant tumour examination, early diagnosis, prognosis evaluation, wind The index of danger assessment, state of illness monitoring and/or curative effect evaluation.

Gal ratios can be calculated using different calculation formula as needed, such as the formula may be selected from：

Formula 1：Gal ratios=Gal₀/(Gal₁+Gal₂)；Or

Formula 2：Gal ratios=Gal₀/(Gal₁+2Gal₂)；Or

It can show that galactolipin (Gal is removed in the end of object blood IgG surfaces double antenna complexity N sugar chains₀) sugared with galactolipin Chain (Gal₁And Gal₂) abundance ratio any other formula, such as it is reciprocal, logarithm other variations.

It is worth noting that, the above-mentioned Gal for abundance ratio₀、Gal₁And Gal₂It is preferred that derive from same sample, same Secondary detection and/or same spectrogram.Therefore, can be avoided using the index caused by the operating process such as the pretreatment of parallel sample partially Difference, so as to reduce error when parallel samples enters Mass Spectrometer Method, it is ensured that the high reappearance and accuracy of analysis, it is ensured that the party Method can be applied to clinical tumor examination, prognosis evaluation, risk assessment, state of illness monitoring and/or curative effect evaluation.

Those of ordinary skill in the art can be drawn corresponding based on common sense in the field according to the detection data of different groups of objects ROC curve, corresponding ROC curve is drawn according to the data of different crowd in embodiment of the invention.It is public according to this area Know technology, each ROC curve both provides a series of threshold values and corresponding sensitivity and specificity.Therefore, use These ROC curves, those skilled in the art can easily find this detection pair when choosing any threshold (i.e. cut-off values) The recognition capability of disease, that is, diagnose sensitivity, specificity and the coincidence rate of corresponding cancer kind.

The calculating (calculating of threshold value) of screening results depends on specificity and sensitivity.The computational methods of threshold value are included but not It is limited to：

Method 1：Maximum slope point in selected ROC curve, it is threshold value to take its corresponding Gal ratio.

Method 2：The value of maximum sensitivity and specificity, i.e. [sensitivity %- (1- specificity %)]_maxCorresponding Gal ratios As optimal threshold (maximum youden index, Youden index).

Using threshold value as boundary, it is determined as the positive higher than the value, is determined as feminine gender less than the value.

Preferable threshold range is 0.35~0.6, preferably 0.5 in the present invention.It will be understood by those skilled in the art that by In test population and the difference of knurl kind, the parameter such as the selection of threshold value and its corresponding sensitivity, specificity and coincidence rate can be Difference, but be all contained in the threshold range that the present invention limits.Those skilled in the art can be such as right according to actual use demand The requirement of specific knurl kind, sensitivity, specificity etc., chosen out of ROC curve disclosed by the invention or threshold range appropriate excellent Select threshold value.Can be it is determined that after optimal threshold, by the scope of optimal threshold ± 1~15% (including any number in the range of this Point or subrange).

Applications of the Gal than index

Gal of the present invention can be applied to malignant tumour examination, early diagnosis, prognosis evaluation, risk assessment, state of an illness prison than index In the purposes such as survey and/or curative effect evaluation.These applications include but is not limited to：

1) Gal is detected than index applied to tumor screening：When Gal ratios reach at or above threshold value set in advance, then sentence The fixed subject is with malignant tumour or with the risk for suffering from malignant tumour.

2) Gal is applied into good dislike than index to judge：When Gal ratios reach at or above threshold value set in advance, then judging should Subject is with malignant tumour or with the risk for suffering from malignant tumour.

3) Gal is applied to Prognosis scoveillance than index：When Gal ratios reach at or above threshold value set in advance, then prompting pair As Malignant Tumor Recurrence or with the excessive risk recurred；

4) Gal is expected than index for high risk group malignant tumour：When Gal ratios reach at or above it is set in advance Threshold value, then prompt object that the possibility of malignant tumour occurs and be higher than other objects；

5) Gal is used for cancer patient's state of illness monitoring than index：When Gal higher than the Gal ratios obtained by previous test than reaching Or higher than scope set in advance (such as 5%~50% or the arbitrfary point in the range of this or subrange), then show that the cancer is suffered from The state of an illness of person has developed or further deteriorated；

6) Gal is used for curative effect evaluation than index：Different time points after before the treatment or in therapeutic process are determined and counted Gal ratios are calculated, when Gal ratios reach at or above scope set in advance (such as 5% higher than the Gal ratios obtained by previous test ~50% or should in the range of arbitrfary point or subrange), then the effect of showing the therapy and/or medicine, is bad.

Method and product

Based on foregoing technology, provided in the present invention with malignant tumour examination, early diagnosis, prognosis evaluation, wind The method and product (such as kit, equipment and/or system) of danger assessment, state of illness monitoring and/or curative effect evaluation.

The method of the present invention comprises the following steps：

Step (C') may include in different application：

1) it is used to tumor screening detect：When Gal ratios reach at or above threshold value set in advance, then judge that the subject suffers from Malignant tumour has the risk for suffering from malignant tumour.

2) it is used to good dislike judge：When Gal ratios reach at or above threshold value set in advance, then judge the subject with pernicious Tumour has the risk for suffering from malignant tumour.

3) it is used for Prognosis scoveillance：When Gal ratios reach at or above threshold value set in advance, then object Malignant Tumor Recurrence is prompted Or the excessive risk with recurrence；

4) it is expected for groups of objects (such as high risk group) malignant tumour：Preset when Gal ratios reach at or above Threshold value, then prompt object occur malignant tumour possibility be higher than other objects；

5) it is used for cancer patient's state of illness monitoring：When Gal higher than the Gal ratios obtained by previous test than reaching at or above advance set Fixed scope (such as 5%~50% or the arbitrfary point in the range of this or subrange), then shown the state of an illness of the cancer patient Development further deteriorates；

6) it is used for curative effect evaluation：Different time points after before the treatment or in therapeutic process determine and calculate Gal ratios, when Gal ratios reach at or above scope set in advance (such as 5%~50% or the model higher than the Gal ratios obtained by previous test Arbitrfary point or subrange in enclosing), then the effect of showing the therapy and/or medicine, is bad.

Threshold range in the inventive method can be 0.35~0.6, preferably 0.5.Can be according to test population and knurl kind not Be adjusted with to threshold range, for example, based on maximum slope point in ROC curve or maximum sensitivity and the value of specificity come It is determined that corresponding threshold range.

In addition, the method for the present invention also optionally includes the one or more steps being selected from the group：

(a') gather and/or deal with objects blood sample；

(b') separation and/or purified blood serum and/or blood plasma；

(c') separation and/or purified blood serum/blood plasma IgG；

(f') judgment threshold is provided；

(h') diagnosis and/or testing result and/or report are provided.

Wherein, can be carried out as follows according to different application, the judgement of step (h')：

(i') it is used to tumor screening detect：When Gal ratios reach at or above threshold value set in advance, then judge that the subject suffers from There is malignant tumour or with the risk for suffering from malignant tumour；

(ii') it is used to good dislike judge：When Gal ratios reach at or above threshold value set in advance, then judge that the subject suffers from Malignant tumour has the risk for suffering from malignant tumour；

(iii') it is used for Prognosis scoveillance：When Gal ratios reach at or above threshold value set in advance, then object malignant tumour is prompted Recurrence or the excessive risk with recurrence；

(iv') it is expected for groups of objects (such as high risk group) malignant tumour：When Gal ratios reach at or above advance set Fixed threshold value, then prompt object that the possibility of malignant tumour occurs and be higher than other objects；

(v') it is used for cancer patient's state of illness monitoring：When Gal is more advance than being reached at or above higher than the Gal ratios obtained by previous test The scope (such as 5%~50% or the arbitrfary point in the range of this or subrange) of setting, then showing the state of an illness of the cancer patient has Develop or further deteriorate；

(vi') it is used for curative effect evaluation：Different time points after before the treatment or in therapeutic process determine and calculate Gal ratios Value, when Gal ratios higher than the Gal ratios obtained by previous test reach at or above scope set in advance (such as 5%~50% or Arbitrfary point or subrange in the range of this), then the effect of showing the therapy and/or medicine, is bad.

Wherein, the threshold value in (i')~(iv') can be 0.35~0.6, preferably 0.5.Those of ordinary skill in the art also may be used Threshold value is set according to specific needs.

The method of the present invention is preferably high-flux detection method, such as can handle 96,192,288,384,480,576 simultaneously Individual sample.

Accordingly, a kind of malignant tumour examination, early diagnosis, prognosis evaluation, risk assessment, disease are additionally provided in the present invention Feelings monitor and/or the product of curative effect evaluation.The product can be, such as kit, equipment, system and/or combinations thereof.Institute Stating product includes：

(A) be used to determining blood IgG surfaces double antenna complexity N sugar chains the horizontal reagent of terminal galactosylation and/ Or instrument, it is used to determine the horizontal Gal of double antenna complexity N- sugar chains that IgG face extremities connect without galactolipin₀, end is connected with The horizontal Gal of double antenna complexity N- sugar chains of 1 galactolipin₁The double antenna complexity N- sugar chains of 2 galactolipins are connected with end Horizontal Gal₂；

(B) optionally, for calculating Gal₀With Gal₁And Gal₂Abundance ratio module or processor；

In addition, the product of the present invention optionally includes the one or more being selected from the group：

(a) it is used for the reagent and/or instrument for gathering and/or dealing with objects blood sample；

(b) it is used for separation and/or the reagent and/or instrument of purified blood serum and/or blood plasma；

(c) it is used for the reagent and/or instrument of separation and/or purified blood serum/blood plasma IgG；

(d) it is used for the reagent and/or instrument for separating, purifying and/or being enriched with serum IgG surface N- sugar chains；

(e) the double antenna complexity N sugar-chain ends for being used to store and/or deal with objects serum remove galactolipin and galactolipin sugar Database, module or processor of the chain abundance ratio as IgG surfaces glycosyl galactose quantitative change index；

(f) it is used for the module or processor that judgment threshold is provided；

(g) it is used for the Gal ratios of object and compares to judge whether object suffers from malignant tumour, object is carried out in advance Assess and/or suffer from afterwards the module or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation；

(h) it is used to provide diagnosis and/or testing result and/or the module or processor of report；

(j) specification or guide for use, wherein describing following one or more applications and judgment mode：

(vi) it is used for curative effect evaluation：Different time points after before the treatment or in therapeutic process determine and calculate Gal ratios, When Gal ratios reach at or above scope set in advance (such as 5%~50% or should higher than the Gal ratios obtained by previous test In the range of arbitrfary point or subrange), then the effect of showing the therapy and/or medicine, is bad.

Wherein, the threshold value in (i)~(iv) can be 0.35~0.6, preferably 0.5.Those of ordinary skill in the art also can root According to specifically needing to set threshold value.

Advantages of the present invention

Main advantages of the present invention are：

1) it is applied to the examination of various malignant tumours；

2) certain specific tumors is directed to, compared with existing clinical diagnosis classical standard, there is specificity height, sensitivity Feature high, the degree of accuracy is high；

3) it is combined with existing clinical diagnosis classical standard, there is higher recall rate；

4) it is convenient to detect, and takes short；For single sample, detected value is reproducible, good stability；High flux inspection can be carried out Survey.

The present invention meets the operation requirement that outstanding clinical diagnosis index should meet during actually detected, shows to detect blood Liquid IgG Gal is than having the reality of clinical practice in malignant tumour examination, early diagnosis, prognosis evaluation, risk assessment etc. Feasibility.

Embodiment

With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention Rather than limitation the scope of the present invention.Those skilled in the art can make appropriate modification to the present invention, change, these modifications It is within the scope of the present invention with variation.

The experimental method of unreceipted actual conditions in the following example, the conventional method in this area can be used, such as joined Examine《Molecular Cloning:A Laboratory guide》(third edition, New York, CSH Press, New York：Cold Spring Harbor Laboratory Press, 1989) or according to the condition proposed by supplier.DNA sequence measurement is that this area is normal The method of rule, also it can provide test by commercial company.

Unless otherwise indicated, otherwise percentage and number are calculated by weight.Unless otherwise defined, it is all used in text Specialty is identical with meaning known to one skilled in the art with scientific words.It is in addition, any similar or equal to described content Deng method and material all can be applied in the inventive method.Preferable implementation described in text only presents a demonstration it with material With.

General introduction

In following experiment of the invention, following sample as shown in table 1 is examined with the method and index of the present invention Survey：

1) (including Healthy People sample+benign check sample, i.e. non-evil sample in table 3A are therein for 924 check samples Benign control is 404 as shown in table 3A)；

2) 1737 preoperative blood samples of malignant tumour (including liver cancer, stomach cancer, lung cancer, oophoroma, intestinal cancer, the cancer of the esophagus, pancreas Gland cancer, cholangiocarcinoma, kidney, breast cancer, prostate cancer, nasopharyngeal carcinoma, cervical carcinoma, carcinoma of urinary bladder, carcinoma of endometrium)；

3) 78 postoperative blood samples of malignant tumour (stomach cancer)；

4) blood sample of 4217 people at highest risk.

Judge that the present invention has high degree of specificity and sensitivity in malignant tumour examination, so as to judge the method for the present invention There is clinical meaning with index.

The sample classification of table 1. and sample age distribution

Materials and methods

I. the source of blood serum sample, classification, pathological data collection, preservation and operation

Samples sources：

Healthy human blood's sample, benign control blood sample, the preoperative blood sample of malignant tumour, precancerous lesion blood samples of patients Sample and the postoperative blood sample of malignant tumour be taken from Grade III Class A hospital be in hospital and out-patient.

Sample classification：

Malignant tumour and precancerous lesion sample：Tumour is suffered from by clear and definite pathological diagnosis or has clear and definite precancerous lesion sufferer Serum sample；Wherein, tumor sample is by stages and hierarchical statistics are shown in Table 2, and statistic of classification is shown in Table 3, and the standard of institute's reference is as described below (quoted from《Malignant tumour INM classification》Handbook).

Commonly use TNM stage standard in the world at present.TNM classification method is to carry out pathology point to cancer according to clinical findings Phase：

The size of 1.T --- primary tumo(u)r (cancer initially occurred), Infiltrating extrent, transfer, infiltration are whether there is as deep as degree, It is divided into (T0 to T4) five grades, the bigger expression cancer progression of numeral must be more obvious, and the different organs occurred according to cancer are formulated Sorting technique be also not quite similar.

The metastasis degree of 2.N --- peripheral lymph node, it is divided into (N0 to N3) four grades, the bigger expression cancer progression of numeral Must be more obvious.

3.M --- DISTANT METASTASES IN situation, M0 represent that M1 then represents to have occurred that other without other organs transfer occurs Organ relative weight.

The I phases：At tumour limitation one, sign is not spread.The II phases：Tumour has spread to neighbouring lymph node, but does not have ripple And other organ or tissues.The III phases：Tumour also involves organ or tissue nearby except being diffused into neighbouring outside lymph node.The IV phases： Tumour has spread to the position of distant place.Its period is more early, and therapeutic effect is better, and the survival of patients time is longer.If implement operation Method cuts off cancer, the cancerous tissue sample for also wanting detailed inspection to cut off after surgery, postoperative TNM stage is carried out to it.

According to cancer cell differentiation degree pathological grading can be carried out to cancer：

1. I grade is to break up, grade malignancy is low；

2.II levels are medium, the grade malignancy moderate of differentiation；

3.III levels are poor to break up, and grade malignancy is high.

The grade malignancy of the pathological grading prompting tumour of malignant tumour.

Healthy People sample：It is normal without any of benign and malignant disease, appearance, without any visible disease symptomses People.

Benign check sample：Reported through clinical biochemical, image or histopathology and make a definite diagnosis benign disease and non-malignant tumors are suffered from The sample of person.

Malignant tumor patient sample：The sample of malignant tumor patient is diagnosed as through histopathology report.

People at highest risk's sample：Refer to the sample for the crowd for having tumorigenic highly dangerous degree.In the people at highest risk of tumour Tumorigenic possibility is significantly larger than population.By taking lung cancer as an example, the male senile patient of smoking for many years is people at highest risk；Again Such as liver cancer, Hepatitis B Virus Infection is people at highest risk.

Sample preservation：5mL venous blood is extracted, (promotees to stand 30 minutes in solidifying pipe) centrifugation of 2000g rotating speeds after solidification under normal temperature 10 minutes, suction out upper serum, -80 DEG C freeze it is standby.

Detection operation：Virus monitory and data processing are implemented under double-blind conditions.

The malignant tumour sample of table 2. is by stages and hierarchical statistics

By stages	Number of cases
		Ⅰ	231
Ⅱ	522
		Ⅲ	603
Ⅳ	196
		Not available	185

		Classification	Number of cases
1	611
		2	246
3	707
		Not available	173

* not available refer to tumor patient by stages and classification not can determine that or look into and takes.

II. instrument and reagent

1.IgG purifying instrument and reagent

Purification column：IgG screenings with albumin A centrifuge plate (Protein A Spin Plate for IgG Screening, 96 Hole) it is purchased from Thermo Fisher Scientific companies, article No. 45202.

Thermo Fisher Scientific public affairs are purchased from reference to cushioning liquid, elution cushioning liquid and BCA kits Department.

2. sugar chain separates and purifying instrument and reagent

Glycosidase：Glycosidase PNGase F (500U/ μ L) are purchased from New England Biolabs companies

Porous graphite carbon PGC：Purchased from Grace companies.

The acetonitrile solution of 0.1% trifluoracetic acid 80%：By acetonitrile:Water:Trifluoracetic acid=80:19.9:0.1 ratio Prepare, the ratio is volume ratio.

0.1% trifluoroacetic acid aqueous solution：By water:Trifluoracetic acid=99.9:0.1 ratio is prepared, and the ratio is body Product ratio.

0.05% acetonitrile solution of trifluoracetic acid 25%：By acetonitrile:Water:Trifluoracetic acid=25:74.95:0.05 ratio Example is prepared, and the ratio is volume ratio.

3. mass spectrum quantitatively uses instrument and reagent

Mass spectrograph：Axima Resonance MALDI-QIT-TOF mass spectrographs (Shimadzu companies).

Uniform reagent：Ethanol, HPLC levels (Merck companies).

Matrix：DHB DHB, purchased from Sigma-Aldrich companies.

Calibration solution standard items：TOFmix, purchased from LaserBio Laboratories companies.

III. method

(I) IgG purification from the blood sample of examination object

Used with reference to Thermo Fisher Scientific Protein A Spin Plate for IgG Screening Family handbook is implemented, specific as follows：

1. purification column and cushioning liquid are balanced 30 minutes at room temperature (with reference to cushioning liquid and elution cushioning liquid)；

2. removing the lid of purifying column bottom, place it on washing plate, the lid at the top of purification column is removed, each 200 μ l combination buffers balance is added in hole；

3. device 1000g rotating speeds assembled above are centrifuged 1 minute；Repeat step (2) and (3) each 1 time；

4. mixing 10 μ l serum and 10 μ l combination cushioning liquid, add in the hole of purification column；

5. slowly concussion is incubated purification column 30 minutes on oscillator；

6. 1000g centrifugal purifications post 1 minute, collecting sample solution (being placed in 96 orifice plates), loading is repeated 1~2 time；

7. purification column is placed on washing/collecting board, 500 μ l combination cushioning liquid are added in every hole, 1000g centrifuges 1 point Clock, this step are repeated 3 times；

8. adding 20 μ l combination cushioning liquid in each collecting board, purification column is placed on collecting board, and in each of which hole Middle addition 200-400 μ l elute cushioning liquid, and slowly concussion is incubated 1 minute, is centrifuged 1 minute with 1000g rotating speed, repeat this step Rapid 2 times；Detected with BCA kits and collect sample, determined the collection liquid residing for IgG, collect the IgG albumen being purified into；

9. adding 400 μ l elution cushioning liquid in each hole of purification column, wash 3 times, then with 400 μ l's 0.02% Sodium azide washs three times, makes purifying column regeneration；

10. adding 100 μ l sodium azide in each hole of purification column or with reference to cushioning liquid, close the lid, being put into can 4 DEG C of preservations in the sack of sealing.

(II) separation and purifying of sugar chain

A. by glucosides ferment treatment, dissociate sugar chain from IgG

1. with 70% ethanol wash 96 orifice plates once in advance, then with milli-Q water 2 times；

2. the IgG sample solutions and glycosidase (New England Biolabs companies, 0705s) of purifying are mixed, 200：1 (v/v)；Above-mentioned mixed liquor adds 96 orifice plates (Corning, 3504), and each sample sets 3 multiple holes；37 DEG C of reactions, 12 hours free sugars Chain.

B. porous graphite carbon (PGC) solid phase extraction concentration purifying sugar chain

1. 96 orifice plates of the activation graphitiferous carbon containing 0.1% trifluoroacetic 80% acetonitrile solution；

2. balance each hole with 0.1% trifluoroacetic acid aqueous solution；

4. deionized water is added in 96 orifice plates of enzyme digestion reaction, per the μ l of hole 150.Then by the 96 of this plate and graphitiferous carbon Orifice plate assembles, and 1000g centrifugations, is repeated 3 times；

5. 100 μ l deionized waters wash impurity and salt, repeated washing 2 times；

6. with the trifluoroacetic 25% acetonitrile solution elution sugar chain for containing 0.05% and collect elution solution, inspection to be analyzed Survey.

(III) the mass spectrum quantitative analysis of sugar chain

Taking the 1 μ l N- sugar chains solution purified in (II) and 1 μ l matrix, (DHB, 10mg/ml are dissolved in ACN/H₂O/TFA (50/50/0.1, v/v/v)) put successively on mass spectrum target plate, dry at room temperature, then it is molten homogeneous with ethanol weight Change, MALDI Mass Spectrometer Method sugar chains.Mass spectral analysis is using TOFMix as mass calibration standard items.

MALDI mass spectroscopy conditions：Using cation reflective-mode；Use 337nm nitrogen lasers source；Laser energy is set It is set to 125V；Sample collection pattern is " auto experiment "；Every MS spectrogram is accumulative 200profiles, 2shot/ profile；It is 500-3000 to gather m/z scopes.

The determination method of three types glycosyl galactose sugar chain signal：In mass spectrogram, the sugar chain of different glycosyl galactoses (Gal₀、Gal₁、Gal₂) there are different mass-to-charge ratioes (m/z), show as different peak (see Fig. 1).Gal in Fig. 1₀Peak appears in m/z At 1485, Gal₁Peak is at m/z 1647, Gal₂Peak is at m/z 1809.

The determination methods of glycosyl galactose degree：Examination ring polymer IgG surfaces double antenna is answered using MALDI mass-spectrometric techniques Miscellaneous type N sugar-chain end glycosyl galactoses degree carries out quantitative analysis comparison, and the double of serum are calculated according to the size of peak height or peak area Antenna complex type N sugar-chain ends remove galactolipin and galactolipin sugar chain abundance ratio (Gal ratio, Gal ratio), and in this, as IgG surfaces glycosyl galactose quantitative change index.Calculation formula used is in following examples：

Gal ratios=[Gal₀/(Gal₁+2*Gal₂)]

Embodiment 1.Gal is than screening/examination for malignant tumour

Using material as described above and method measure control serum samples (n=924) and sera of patients with malignant tumors sample IgG glycosyl galactose degree in product (n=1737).Testing result is as follows：

1. the MS spectrograms of 2661 serum IgG N- sugar chains are amounted to by observing, it can be found that Gal ratios are suffered from malignant tumour With notable difference be present between compareing in person.Representative spectrogram is as shown in Figure 1.

2. pair 2661 sample Gal ratios are compared, while carry out Receiver operating curve (receiver Operating characteristic curve, ROC curve) analysis.As a result show：Tumor screening index is used for Gal, AUC is 0.9385 (95% confidential interval：0.9125~0.9608) it is corresponding special, in the case where controlling sensitivity to be 90% Degree is up to 88% (see Fig. 2A).With reference to AUC criterion (as follows), this index is used as diagnosis index pin-point accuracy.

AUC criterion (Clin.Chem.2007,53：1615-22.)：ROC curve is usually used in comparing sentencing in clinic Recognition capability of the disconnected diagnosis index to disease.In theory, as AUC (area-under-the-curve) >=0.9, the diagnosis refers to Mark is considered as " pin-point accuracy "；When AUC is between 0.7-0.9 and 0.5-0.7, respectively " applicable " and engineering noise.

3. numerically, the Gal ratios of malignant tumor patient and the extremely notable (p value of check sample difference<0.0001) (see Table 3A).

4. simultaneously, using threshold value 0.5 (hereafter judging also to be based on the threshold value done in other embodiments) as judge should be by Standard of the examination person with malignant tumour or with the risk for suffering from malignant tumour, as a result finds (being shown in Table 3B)：

1) in above-mentioned sample, malignant tumour recall rate is up to 88.72% or so；

2) to the recall rate of certain specific tumors also apparently higher than existing classical index, such as：

In liver cancer case, using Gal ratios and AFP as independent diagnostics index, recall rate is respectively 93.98% He 59.72%, AFP negative (<87 liver cancer serum samples 20ng/ml) are detected, and the inventive method can detect 72；

In oophoroma, using Gal ratios and CA125 as independent diagnostics index, recall rate is respectively 92.34% He 69.79%, CA-125 it is positive (>In 249 gynecological benign diseases patients serums 35U/ml), the inventive method can detect 218.

Table 3A Gal are than the statistical form in malignant tumor patient and check sample

* non-evil sample：For Healthy People+benign control

**P<0.0001 shows there is statistically extremely significant difference

Table 3B. stagings and various index recall rates compare

Note：Other cancers include breast cancer, nasopharyngeal carcinoma, cervical carcinoma, carcinoma of urinary bladder, carcinoma of endometrium.

Joint Index refers to：The Gal of the present invention is combined for examining than index with existing classical index

Result above shows, using Gal than energy high sensitivity, the sieve effectively utilized in malignant tumour of high specific Look into, compared with existing classical standard, method of the invention and index have higher accuracy.Also, by the method for the present invention The recall rate that can further improve malignant tumour is used in combination with index and existing classical standard.

Embodiment 2.Gal ratios are used to distinguish ovarian cancer patients and compare (health and benign disease)

Sample (n=235) obtained from ovarian cancer patients is with compareing AUC=0.9486 (95% confidence areas obtained by (n=924) Between：0.9329,0.9644), referring to Fig. 2 B.

Than average, extremely significantly (t is examined difference the Gal of ovarian cancer patients, p compared with check sample<0.0001).(referring to Table 3A).

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish ovarian cancer patients and control.

Embodiment 3.Gal ratios are used to distinguish patients with lung cancer with compareing

Sample (n=206) obtained from patients with lung cancer is with compareing AUC=obtained by (Healthy People and benign disease) (n=924) 0.9578 (95% confidential interval：0.9433,0.9724), referring to Fig. 2 C.

Than average, extremely significantly (t is examined difference the Gal of patients with lung cancer, p compared with check sample<0.0001) (referring to table 3A)。

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish patients with lung cancer with compareing.

Embodiment 4.Gal ratios are used to distinguish patients with gastric cancer with compareing

Sample (n=158) obtained from patients with gastric cancer is with compareing AUC=obtained by (Healthy People and benign disease) (n=924) 0.9477 (95% confidential interval：0.9304,0.9650), referring to Fig. 2 D.

Than average, extremely significantly (t is examined difference the Gal of patients with gastric cancer, p compared with check sample<0.0001) (referring to table 3A)。

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish stomach cancer and control.

Embodiment 5.Gal ratios are used to distinguish liver cancer patient with compareing

Sample (n=216) obtained from liver cancer patient is with compareing (health and benign disease) AUC=obtained by (n=924) 0.9497 (95% confidential interval：0.9327,0.9667), referring to Fig. 2 E.

Than average, extremely significantly (t is examined difference the Gal of liver cancer patient, p compared with check sample<0.0001) (referring to table 3A)。

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish liver cancer and control.

Embodiment 6.Gal ratios are used to distinguish patients with bowel cancer with compareing

Sample (n=204) obtained from patients with bowel cancer is with compareing (health and benign disease) AUC=obtained by (n=924) 0.9468 (95% confidential interval：0.9289,0.9647), referring to Fig. 2 F.

Than average, extremely significantly (t is examined difference the Gal of patients with bowel cancer, p compared with check sample<0.0001) (referring to table 3A)。

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish intestinal cancer and control.

Embodiment 7.Gal ratios compare for distinguishing all kinds of malignant tumours with benign

Sample (n=1737) obtained from malignant tumor patient and AUC=0.9099 obtained by benign disease sample (n=404) (95% confidential interval：0.8924~0.9275) (referring to Fig. 2 G).

Than average, extremely significantly (t is examined difference the Gal of malignant tumor patient, p compared with check sample<0.0001) (ginseng It is shown in Table 3A).

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish malignant tumor patient and benign control.

Embodiment 8.Gal has higher diagnostic value than being used in combination with classical index

As described in table 3B, when Gal after classical index conjunctive use than with that can improve the recall rate in specific tumors. In 216 liver cancer patients, Gal ratios and AFP conjunctive uses, recall rate, which reaches 95.83%, (has a positive to sentence in two indices It is set to the positive), independently detected higher than Gal ratios (93.98%) and AFP (59.72%)；In 158 patients with gastric cancer, Gal Than reaching 91.14% with CEA conjunctive uses, recall rate, independently examined higher than Gal ratios (88.61%) and CEA (59.49%) Survey.

Summary data, Gal ratios can be used in combination with classical index, increase malignant tumour recall rate.

Embodiment 9.Gal ratios are applied to early diagnosis

Sample (n=231) obtained from primary malignant tumor patient (neoplasm staging is I i.e. in table 2) is (healthy and good with compareing Property disease) AUC=0.8958 (95% confidential intervals obtained by (n=924)：0.8724,0.9192), referring to Fig. 2 H.

Than average, extremely significantly (t is examined difference the Gal of primary malignant tumor patient, p compared with check sample< 0.0001)。

Above-mentioned analysis confirms that the serum IgG glycosyl galactose index of the present invention can effectively be sieved with high sensitivity and specificity Look into and distinguish primary malignant tumour and control.

Embodiment 10.Gal ratios are applied to monitoring tumor prognosis

The patients with gastric cancer follow-up visit monitoring of 78 customary specification operation plan treatments is studied, to examine Gal ratios and trouble in blood The correlation of person's change of illness state, detection cycle be every 3 months once.

The recurrence of postoperative tumour is evaluated according to pathological diagnosis, clinical biochemical and imaging data.As a result show, In 3 years after surgery, 25 recurrences are shared, wherein before the diagnosis of clear and definite tumor recurrence, there are 23 Gal than positive (recall rate For 92.0%).

Data above demonstrates the inventive method has important value in the prognosis detection of tumour.

Embodiment 11.Gal is than the generation for predicting malignant tumour

Be monitored for the serum sample of 4217 people at highest risk, and carry out effective tracking not etc. in 6 months to 3 years with Visit, Follow-up results find that than being found in 3780 people of negative group without tumor patient, tumour patient 47 is had found in the positive 437 people of group by Gal Example, two groups of Tumor incidence significant differences.

Accordingly, Gal can be used for the generation of prediction malignant tumour than detecting.

All it is incorporated as referring in this application in all documents that the present invention refers to, it is independent just as each document It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited Enclose.

Claims

1. one kind is commented for the examination of object malignant tumour, early diagnosis, prognosis evaluation, risk assessment, state of illness monitoring and/or curative effect The product estimated, the product are kit, equipment, operable system and/or combinations thereof, and the product includes：

(A) be used to determining the reagents of terminal galactosylation degree of blood IgG surfaces double antenna complexity N sugar chains, instrument and/ Or system, it is used to determine the double antenna complexity N- sugar chain abundance Gal that IgG face extremities connect without galactolipin₀, end is connected with The double antenna complexity N- sugar chain abundance Gal of 1 galactolipin₁The double antenna complexity N- sugar chains of 2 galactolipins are connected with end Abundance Gal₂；

(C) optionally, for according to Gal than judging whether object suffers from malignant tumour, object progress and/or is suffered from prognosis evaluation Suffer from the module and/or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation；

Wherein, the malignant tumour is selected from：Liver cancer, stomach cancer, lung cancer, intestinal cancer, the cancer of the esophagus, cancer of pancreas, cholangiocarcinoma, kidney, mammary gland Cancer, prostate cancer, nasopharyngeal carcinoma, carcinoma of urinary bladder, carcinoma of endometrium and cervical carcinoma.

2. product as claimed in claim 1, it is characterised in that the blood is selected from：Serum, blood plasma and whole blood.

3. product as claimed in claim 1, it is characterised in that being answered for determining blood IgG surfaces double antenna in (A) Reagent, instrument and/or the system of the terminal galactosylation degree of miscellaneous type N sugar chains are the one or more sides for being selected from the group Reagent, instrument and/or the system of method：MALDI TOF MS method MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, electrospray ionization mass spectrum ES-MS；Liquid chromatography；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip technology；Nuclear magnetic resonance nmr.

4. product as claimed in claim 1, it is characterised in that the product also includes the one or more being selected from the group：

E) the double antenna complexity N sugar-chain ends for being used to store and/or deal with objects serum go galactolipin and galactolipin sugar chain rich Spend database, module and/or processor of the ratio as IgG surfaces glycosyl galactose quantitative change index；

G) it is used to comparing comment the Gal ratios of object to judge whether object suffers from malignant tumour, carries out object prognosis Estimate and/or suffer from the module and/or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation；

(i) it is used to tumor screening detect：When Gal ratios reach at or above threshold value set in advance, then judge the subject with disliking Property tumour or with suffering from the risk of malignant tumour；

(ii) it is used to good dislike judge：When Gal ratios reach at or above threshold value set in advance, then judge the subject with pernicious swollen Knurl has the risk for suffering from malignant tumour；

(iii) it is used for Prognosis scoveillance：When Gal ratios reach at or above threshold value set in advance, then object Malignant Tumor Recurrence is prompted Or the excessive risk with recurrence；

(iv) it is expected for groups of objects malignant tumour：When Gal ratios reach at or above threshold value set in advance, then object is prompted The possibility that malignant tumour occurs is higher than other objects；

(v) it is used for cancer patient's state of illness monitoring：When Gal is preset than being reached at or above higher than the Gal ratios obtained by previous test Scope, then show that the state of an illness of the cancer patient has developed or further deteriorated；

(vi) it is used for curative effect evaluation：Different time points after before the treatment or in therapeutic process determine and calculate Gal ratios, when Gal ratios reach at or above scope set in advance higher than the Gal ratios obtained by previous test, then show the therapy and/or medicine The effect of thing, is bad.

5. product as claimed in claim 4, it is characterised in that it is described d) in be used for separate, purify and/or be enriched with serum The reagent and/or instrument of IgG surfaces N- sugar chains are that what is be selected from the group isolate and purify the reagent and/or instrument of N sugar chain methods：It is porous Graphitized carbon PGC solid phase extractions, polysaccharide purification post, agglutinin affinity method, Capillary Electrophoresis, high performance liquid chromatography.

6. product as claimed in claim 4, it is characterised in that the threshold value set in advance in (i)~(iv) is by optimal threshold ± 1%~15% determines, wherein optimal threshold is based on maximum slope point or maximum sensitivity in ROC curve and specificity Value determines.

7. product as claimed in claim 4, it is characterised in that threshold value set in advance in (i)~(iv) for 0.35~ 0.6。

8. product as claimed in claim 4, it is characterised in that in (v), when Gal is than the Gal obtained by higher than previous test Than reaching at or above 5%~50%, then show that the state of an illness of the cancer patient has developed or further deteriorated.

9. product as claimed in claim 4, it is characterised in that (vi) be used for curative effect evaluation in, before the treatment after or control Different time points during treatment determine and calculate Gal ratios, when Gal ratios reach higher than the Gal ratios obtained by previous test Or higher than 5%~50%, then the effect of showing the therapy and/or medicine, is bad.

10. product as claimed in any one of claims 1-9 wherein, it is characterised in that the Gal₀With Gal₁And Gal₂Abundance ratio The calculating of value is carried out using the formula being selected from the group：

Gal ratios=Gal₀/(Gal₁+Gal₂)；

Gal ratios=Gal₀/(Gal₁+2Gal₂)；Or

Its reciprocal, logarithmic transformation form.

11. product as claimed in claim 1, it is characterised in that the product is to contain in the detection method of following steps：

(A') terminal galactosylation of IgG surfaces double antenna complexity N sugar chains is horizontal in measure object blood sample, the end End Galactosylation levels include：The horizontal Gal of double antenna complexity N- sugar chains that IgG face extremities connect without galactolipin₀, end It is connected with the horizontal Gal of double antenna complexity N- sugar chains of 1 galactolipin₁The double antenna complexity N- of 2 galactolipins is connected with end The horizontal Gal of sugar chain₂；

(C') according to Gal than judging whether object suffers from malignant tumour, carry out prognosis evaluation to object and/or suffers from the wind of tumour Assess, the state of an illness is monitored and/or curative effect is assessed in danger.

12. product as claimed in claim 11, it is characterised in that methods described also includes the one or more steps being selected from the group Suddenly：

(a') gather and/or deal with objects blood sample；

(b') separation and/or purified blood serum and/or blood plasma；

(c') separation and/or purified blood serum/blood plasma IgG；

(f') judgment threshold is provided；

(g') by the Gal ratios of object and compare with judge object whether suffer from malignant tumour, to object carry out prognosis evaluation And/or suffer from tumour, state of illness monitoring and/or curative effect evaluation；

(h') diagnosis and/or testing result and/or report are provided；

(j') other malignant tumour indexs are detected, and the testing result of other malignant tumour indexs and Gal are tied than detection Fruit combines.

13. product as claimed in claim 1, it is characterised in that the product also includes to be referred to for detecting other malignant tumours Target kit, equipment, system and/or combinations thereof.

14. product as claimed in claim 13, it is characterised in that the index is selected from：The basic condition of patient, clinical tumor Performance, selectivity physico-chemical examination index.

15. product as claimed in claim 14, it is characterised in that the basic condition of the patient includes one in being selected from the group Kind is a variety of：Age, sex, family history.

16. product as claimed in claim 14, it is characterised in that the clinical tumor shows one kind or more in being selected from the group Kind：Topical manifestations, general manifestation and interior examine；

Wherein, topical manifestations include, lump and secondary symptom, pain, pathological discharge thing, one kind in skin and mucosa ulcer or It is a variety of；

Wherein, general manifestation includes heating, infection, anaemia, becomes thin, be weak, the one or more in dyscrasia；

Wherein, inside examine including the one or more in digital rectal examination, gynecologial examination.

17. product as claimed in claim 14, it is characterised in that the selectivity physico-chemical examination index is included through specific shadow Index as checking gained, wherein, the specific image check be selected from the group in one or more：It is nuclear-magnetism, CT, B ultrasound, interior Sight glass, breast molybdenum target.

18. product as claimed in claim 14, it is characterised in that the selectivity physico-chemical examination index refers to including tumor biochemistry Mark, wherein, the tumor biochemistry index be selected from the group in one or more：AFP、CEA、PSA、SF、TSGF、POA、 PROGRP、CA15-3、CA19-9、CA72-4、CA242、CA50、CYFRA21-1、NSE、SCC、AFU、EBV-VCA。

19. reagent, instrument, module and/or processor prepare for the examination of object malignant tumour, early diagnosis, prognosis evaluation, Application in the product of risk assessment, state of illness monitoring and/or curative effect evaluation, the product be kit, equipment, system and/or Combinations thereof, the reagent, instrument, module and/or processor include：

(A) it is used for reagent, instrument, the mould of terminal galactosylation level for determining blood IgG surfaces double antenna complexity N sugar chains Block and/or processor, it is used to determine the horizontal Gal of double antenna complexity N- sugar chains that IgG face extremities connect without galactolipin₀、 End is connected with the horizontal Gal of double antenna complexity N- sugar chains of 1 galactolipin₁The double antenna that 2 galactolipins are connected with end is complicated The horizontal Gal of type N- sugar chains₂；

(C) optionally, for according to Gal than judging whether object suffers from malignant tumour, object progress and/or is suffered from prognosis evaluation Suffer from the module or processor of the risk assessment of tumour, state of illness monitoring and/or curative effect evaluation,

20. application as claimed in claim 19, it is characterised in that being used in (A) determines blood IgG surfaces double antenna The terminal galactosylations of complexity N sugar chains horizontal reagent, instrument, module and/or processor be for be selected from the group one Reagent, instrument, module and/or the processor of kind or a variety of methods：MALDI TOF MS MALDI methods, Fast atom bombardment mass spectroscopy FAB-MS, electrospray ionization mass spectrum ES-MS；Liquid chromatography；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip skill Art；Nuclear magnetic resonance nmr.

21. application as claimed in claim 19, it is characterised in that the product is as any one of claim 1-18.

22. a kind of method screened for malignant tumor medicine and/or treatment method, methods described include：

(A ") measure object be administered and/or treatment before, afterwards and/or during different time points blood sample in IgG tables The terminal galactosylation of face double antenna complexity N sugar chains is horizontal, and the terminal galactosylation level includes：IgG surfaces end Hold the horizontal Gal of double antenna complexity N- sugar chains without galactolipin connection₀, end be connected with the double antenna complexity N- of 1 galactolipin The horizontal Gal of sugar chain₁The horizontal Gal of double antenna complexity N- sugar chains of 2 galactolipins is connected with end₂；

(B ") calculates Gal₀With Gal₁And Gal₂Abundance ratio, i.e. Gal ratios；

(C ") judges the validity of the medicine and/or treatment method according to Gal than assessing, wherein when Gal ratios are higher than previous The Gal ratios of test gained reach at or above scope set in advance, then the effect of showing the medicine and/or treatment method is not It is good；

23. method as claimed in claim 22, it is characterised in that in (C "), when Gal ratios are higher than obtained by previous test Gal ratios reach at or above 5%~50%, then the effect of showing the medicine and/or treatment method, is bad.