CN106596827A - Use of Gal index in assessment of autoimmune disease treatment sensitivity and efficacy - Google Patents

Use of Gal index in assessment of autoimmune disease treatment sensitivity and efficacy Download PDF

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CN106596827A
CN106596827A CN201710057969.6A CN201710057969A CN106596827A CN 106596827 A CN106596827 A CN 106596827A CN 201710057969 A CN201710057969 A CN 201710057969A CN 106596827 A CN106596827 A CN 106596827A
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gal
indexs
igg
galactose
targeted therapy
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CN106596827B (en
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顾建新
任士芳
孙琳琳
周蕾
张兴旺
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Shanghai Zhixian Biological Technology Co Ltd
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Abstract

The invention provides a use of a Gal index in assessment of autoimmune disease treatment sensitivity and efficacy and relates to a product, use and method for forecasting and screening autoimmune disease treatment sensitivity, monitoring disease conditions and/or assessing curative effects through detecting an immunoglobulin G surface double-antenna complex N-linked oligosaccharide terminal galactose index of blood and its change.

Description

Application of the Gal indexs in treating autoimmune diseases sensitivity and curative effect evaluation
Technical field
The invention belongs to biotechnology and medical domain, are related to cytokine targeting preparation in treatment autoimmunity disease The monitoring of sensitivity screening, the state of an illness and therapeutic effect in disease.More particularly it relates to pass through to determine in blood sample The level of Gal indexs judges that cytokine targeting preparation treatment (such as TNF-α inhibitor for treating, such as Antybody therapy) is exempted from itself The monitoring of the sensitivity in epidemic disease disease treatment and the state of an illness and therapeutic effect to autoimmune disease.
Background technology
It is the chronic disease for causing that autoimmune disease is that the immunologic tolerance of autoantigen damages, but the cause of disease and morbidity machine System is very clear and definite, wherein representational disease is included：Tetanic type spondylitiss (AS), rheumatoid arthritiss (RA), system Property lupus erythematosus (SLE), multiple sclerosiss (MS) etc..These disease courses are long, easily disable, and easily cause all multi viscera diseases Become.Therefore, how to carry out early treatment to reduce the disability rate of patient, improve the life quality of patient, be always the research and lead A domain difficult problem urgently to be resolved hurrily and focus.
The treatment of various autoimmune disease at present without radical cure medicine, clinically relies primarily on immunosuppressant, wraps Containing non-selective immunosuppressant (such as glucocorticoid, alkylating agent, antimetabolite etc.) and selectivity immunosuppressant (as selected Property T cell inhibitor, cytokine and its antagonist, anti-CD molecule monoclonal antibodies etc.).With traditional non-selective immunity suppression Preparation is compared, and selectivity immunosuppressant has relatively low untoward reaction and good curative effect.
At present, the medicine of targeted cytokines TNF-α and interleukin is widely used in clinic.For example, in AS and In the treatment of RA, the TNF-α inhibitor of targeted cytokines TNF-α is applied to clinical more than ten years abroad, at home in formula City also 7 years, is included into medical insurance and achieves preferable curative effect.Especially anti-tnf-alpha monoclonal antibody medicine Embrel, Ying Fu Former times, adalimumab etc., clinical efficacy really, are referred to as the once leap and revolution of AS and RA treatment histories.But, antibody drug Medical expense is expensive, by taking AS as an example, still have more than 30% patient it is invalid Jing after the treatment of above-mentioned antibody medicine, for them, this Plant expensive antibody drug and treat actually useless work(.Therefore, screen to subtracting for patient groups carry out sensitivity before the treatment Light country's individual burden of ineffective investment and patient in terms of medical insurance has long-range meaning.
Thus, this area can be used for the treatment of autoimmune disease cytokine targeting preparation in the urgent need to developing one kind The method of sensitivity screening, to reach the purpose precisely treated and optimize allocation of resources.
The content of the invention
One of main object of the present invention is to overcome the shortcomings of that prior art is present, there is provided a kind of detection based on blood Index Gal index, for the sensitivity screening of autoimmune disease cytokine targeting preparation treatment, to autoimmunity disease The curative effect evaluation and state of illness monitoring of disease.
In one aspect of the invention, there is provided it is a kind of for cytokine targeting preparation in autoimmune disease Therapeutic sensitivity screening, the product of state of illness monitoring and/or curative effect evaluation, the product can be such as test kit, equipment, operable System and/or combinations thereof, the product include：
(A) for determining reagent, the instrument of the terminal galactosylation degree of blood IgG surfaces double antenna complexity N sugar chain Device and/or system, which is used to determine double antenna complexity N- sugar chain abundance Gal that IgG face extremities connect without galactose0And end End is connected with double antenna complexity N- sugar chain abundance Gal of 2 galactose2；
For example, the reagent of one or more method for being selected from the group, instrument and/or system：Ground substance assistant laser solution It is analysis time-of-flight mass spectrometry (TOFMS) MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, Electrospray Mass Spectrometry ESI-MS, liquid chromatography, super High performance liquid chromatography, Liquid Chromatography-Mass Spectrometry, carbohydrate chip technology, microflow control technique, nuclear magnetic resonance nmr, preferred ultra high efficiency Liquid chromatograph；
(B) for calculating Gal0And Gal2Abundance ratio Gal indexs module and/or processor；
(C) it is optional, for judging that object treats whether sensitive, right for cytokine targeting preparation according to Gal indexs Object carries out the module and/or processor of 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, preferred serum.
In some embodiments, the Cytokine Targeted Therapy is directed to TNF-α, IL-1 β (interleukin-1 ' beta ') And/or IL-6 (interleukin-6).
In some embodiments, the Cytokine Targeted Therapy is Antybody therapy.
In certain embodiments of the present invention, the product also includes one or more for being selected from the group：
A) for gathering and/or deal with objects the reagent and/or instrument of blood sample；
B) for separation and/or the reagent and/or instrument of purified blood serum and/or blood plasma；
C) for separation and/or the reagent and/or instrument of purified blood serum/blood plasma IgG；
D) for separating, the reagent and/or instrument of purification and/or enrichment 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 Weighing apparatus PGC, using acetonitrile:Water:Trifluoracetic acid=25:74.95:0.05 mixed solvent eluting sugar chain), polysaccharide purification post, coagulation Plain affinity method (such as continuous agglutinin affinity chromatography SLAC), capillary electrophoresis, high performance liquid chromatography；
E) for the reagent and/or instrument of labelling (such as fluorescent labeling) IgG surfaces N- sugar chains；
F) the double antenna complexity N sugar-chain end for storing and/or dealing with objects serum removes galactose with galactose sugar Chain abundance ratio is used as the data base of IgG surfaces glycosyl galactose quantitative change index, module and/or processor；
G) for providing the module and/or processor of judgment threshold；
H) for according to Gal indexs judge object in the treatment of cytokine targeting preparation it is whether sensitive, disease is carried out to object Feelings monitoring and/or the module and/or processor of curative effect evaluation.
I) for providing the module and/or processor of testing result and/or report；
J) description or guide for use, wherein describing following one or more application and judgment mode：
I () is screened for autoimmune disease sensitivity：When Gal indexs reach at or above threshold value set in advance, then Judge that the autoimmune disease patient is sensitive to the treatment of cytokine targeting preparation；
(ii) for autoimmune disease patient's state of illness monitoring：When Gal indexs are higher than the Gal indexs obtained by previous test Scope set in advance (such as 5%～50% or the arbitrfary point in the range of this or subrange) is reached at or above, then shows this certainly The state of an illness of body immune disease patient has developed or has further deteriorated；
(iii) for curative effect evaluation：Different time points after before the treatment or in therapeutic process are determined and calculate Gal and refer to Mark, when Gal indexs higher than the Gal indexs 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 being somebody's turn to do), then show the unsatisfactory curative effect of the therapy and/or medicine；
It is preferred that, the threshold value in (i)～(iii) is 2.2～3.5, more preferably 3.065.
In some embodiments, can be based on the value of maximum slope point or maximum sensitivity and specificity in ROC curve To determine optimal threshold, and preferred threshold range can be that optimal threshold ± 1～15% (includes any number point in the range of this Or subrange) scope.
In certain embodiments of the present invention, the Gal indexs are calculated is carried out using the formula being selected from the group：Gal ratios Value=aGal0/bGal2, or other variations such as its inverse, logarithm.Wherein, the respective value of a, b is separately located in more than 0 To between 10, such as independently selected from 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,1.2,1.5,2,2.5,3, 3.5th, 4,5,6,7,8,9 or 10, wherein a ≠ 0；b≠0.Can be according to various parameters in the test instrunment and/or method for being adopted Gal0And Gal2Responsiveness, the concrete value of a or b is adjusted.For example, can be based on the test method for being used and mark Ratio after quasi- method (such as Ultra Performance Liquid Chromatography, UPLC) verification, is adjusted to the concrete value of a, b.
For example, in certain embodiments of the present invention, the formula being selected from the group can be adopted to carry out ratio calculation：
Gal indexs=Gal0/Gal2；Or other variations such as its inverse, logarithm.
In certain embodiments of the present invention, the autoimmune disease is selected from：Ankylosing spondylitises, rheumatoid Arthritis, arthritic psoriasises, adolescent arthritis, plaque psoriasis, ulcerative colitiss, Crohn ' s diseases, systematicness Lupus erythematosus, multiple sclerosiss, systemic sclerosises, sjogren syndrome.
In certain embodiments of the present invention, described pair as if mammal, preferred people, monkey, Canis familiaris L., horse, cattle, sheep, pig, Mus, rabbit etc..
In certain embodiments of the present invention, the cytokine targeting preparation is predominantly targeting TNF-α, and for example which is optional From：Embrel, Infliximab and adalimumab.It is preferred that Embrel and Infliximab.
In certain embodiments of the present invention, the products application is in the detection method for comprising the steps of：
(A') in measure object blood sample IgG surfaces double antenna complexity N sugar chain terminal galactosylation level, institute Stating terminal galactosylation level includes：The horizontal Gal of double antenna complexity N- sugar chain that IgG face extremities connect without galactose0 The horizontal Gal of double antenna complexity N- sugar chain of 2 galactose is connected with end2；
(B') calculate Gal0And Gal2Abundance ratio, i.e. Gal indexs；
(C') judge whether object is sensitive for the treatment of cytokine targeting preparation, the object state of an illness is entered according to Gal indexs Row is monitored and/or curative effect is estimated.
In some embodiments, the Cytokine Targeted Therapy is directed to TNF-α, IL-1 β (interleukin-1 ' beta ') And/or IL-6 (interleukin-6).
In some embodiments, the Cytokine Targeted Therapy is Antybody therapy.
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') separate and/or purified blood serum and/or blood plasma；
(c') separate and/or purified blood serum/blood plasma IgG；
(d') separation, purification and/or enrichment serum IgG surface N- sugar chains；
(e') the double antenna complexity N sugar-chain end for storing and/or dealing with objects serum removes galactose and galactose sugar chain Abundance ratio is used as IgG surfaces glycosyl galactose quantitative change index；
(f') provide judgment threshold；
(g') by the Gal ratios of object with compare to judge object whether for cytokine targeting preparation treats quick Feel, the object state of an illness is monitored and/or curative effect is estimated.
(h') testing result and/or report are provided；
In another aspect of the present invention, there is provided reagent, instrument, module and/or processor prepare for reagent, Whether instrument, module and/or processor are being prepared for object to Cytokine Targeted Therapy sensitivity, state of illness monitoring and/or treatment Effect assessment product in application, the product can be such as test kit, equipment, system and/or combinations thereof, the examination Agent, instrument, module and/or processor include：
(A) for determining reagent, the instrument of the terminal galactosylation level of blood IgG surfaces double antenna complexity N sugar chain Device, module and/or processor, which is used to determine the double antenna complexity N- sugar chain level that IgG face extremities connect without galactose Gal0, and end be connected with the horizontal Gal of double antenna complexity N- sugar chain of 2 galactose2；
For example, the reagent of one or more method for being selected from the group, instrument, module and/or processor：Matrix-assisted Laser desorption time-of-flight mass spectrometry (TOFMS) MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, Electrospray Mass Spectrometry ESI-MS；Liquid chromatograph Method；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip technology；Microflow control technique；Nuclear magnetic resonance nmr, preferred liquid phase chromatography；
(B) it is optional, for calculating the module or processor of Gal indexs；
(C) it is optional, for whether judging object for Cytokine Targeted Therapy is sensitive according to Gal indexs, to object The module or processor of state of illness monitoring and/or curative effect evaluation.
In certain embodiments of the present invention, the product is as previously mentioned.
In a still further aspect thereof, there is provided a kind of to be used for autoimmune disease patient's cytokine targeting system The method of the agent/screening of Drug therapy sensitivity, autoimmune disease patient's state of illness monitoring and/or curative effect evaluation, its feature exist In methods described employs the application of the product or the present invention of the present invention.
In some embodiments, methods described includes：
(A') in measure object blood sample IgG surfaces double antenna complexity N sugar chain terminal galactosylation level, institute Stating terminal galactosylation level includes：The horizontal Gal of double antenna complexity N- sugar chain that IgG face extremities connect without galactose0 The horizontal Gal of double antenna complexity N- sugar chain of 2 galactose is connected with end2；
(B') calculate Gal0And Gal2Abundance ratio, i.e. Gal indexs；
(C') judge whether object is sensitive to Cytokine Targeted Therapy, object is carried out according to Gal indexs or its change State of illness monitoring and/or curative effect is estimated.
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') separate and/or purified blood serum and/or blood plasma；
(c') separate and/or purified blood serum/blood plasma IgG；
(d') separation, purification and/or enrichment serum IgG surface N- sugar chains；
(e') for the reagent and/or instrument of labelling (such as fluorescent labeling) IgG surfaces N- sugar chains；
(f') the double antenna complexity N sugar-chain end for storing and/or dealing with objects serum removes galactose and galactose sugar chain Abundance ratio is used as IgG surfaces glycosyl galactose quantitative change index；
(g') provide judgment threshold；
(h') the Gal indexs of object are compared with threshold value with judge object it is whether sensitive for Cytokine Targeted Therapy, State of illness monitoring and/or curative effect evaluation are carried out to object；
(i') diagnosis and/or testing result and/or report are provided；
In some embodiments, methods described is used for purposes as described below, and including corresponding steps as described below：
I () is screened for Cytokine Targeted Therapy sensitivity：When Gal indexs reach at or above threshold value set in advance, Then judge that the experimenter is sensitive for Cytokine Targeted Therapy；
(ii) for autoimmune disease patient's state of illness monitoring：When Gal indexs are higher than the Gal indexs obtained by previous test Scope set in advance (such as 5%～50% or the arbitrfary point in the range of this or subrange) is reached at or above, then shows the trouble The state of an illness of person has developed or has further deteriorated；
(iii) for curative effect evaluation：Different time points after before the treatment or in therapeutic process are determined and calculate Gal and refer to Mark, when Gal indexs higher than the Gal indexs 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 being somebody's turn to do), then show the unsatisfactory curative effect of the therapy and/or medicine；
It is preferred that, the threshold value in (i)～(iii) is 2.2～3.5, more preferably 3.065.
In some embodiments, the Cytokine Targeted Therapy is directed to TNF-α, IL-1 β (interleukin-1 ' beta ') And/or IL-6 (interleukin-6).
In some embodiments, the Cytokine Targeted Therapy is Antybody therapy.
In some embodiments, can be based on the value of maximum slope point or maximum sensitivity and specificity in ROC curve To determine optimal threshold, and preferred threshold range can be that optimal threshold ± 1～15% (includes any number point in the range of this Or subrange) scope.
In certain embodiments of the present invention, the product and application is used in the method for comprising the steps：
Separated and/or purified blood serum IgG from ring polymer sample with IgG purification columns, it is preferred to use with purification column kit Alkalescence combine buffer solution and faintly acid drip washing buffer solution；
With the detached IgG of glucosides ferment treatment separating the N sugar chains of IgG, 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, adopts acetonitrile:Water:Trifluoracetic acid=25: 74.95:0.05 mixed solvent eluting sugar chain；
Using Gal in liquid chromatograph quantitative analysis N- sugar chains0And Gal2Level；
Show whether the patient is applied to Cytokine Targeted Therapy based on ring polymer IgG surface Ga l indexs, and to suffering from Person carries out state of illness monitoring and/or curative effect evaluation.
Those skilled in the art can carry out combination in any without deviating from this to aforesaid technical scheme and technical characteristic Bright inventive concept and protection domain.The present invention other side due to this disclosure, to those skilled in the art For be obvious.
The invention will be further described to combine accompanying drawing and subordinate list further below, wherein shown only for illustrating this Bright embodiment, rather than in order to limit to the scope of the present invention.
Description of the drawings
Fig. 1：The ROC curve of quick group of AS patient's TNF-α inhibitor height and TNF-α inhibitor hypoallergenic group Gal Indexes Comparison.
Fig. 2：The ROC curve of quick group of RA patient's TNF-α inhibitor height and TNF-α inhibitor hypoallergenic group Gal Indexes Comparison.
Subordinate list explanation
Table 1：AS patient's TNF-α inhibitor for treating result judgement.
Table 2：AS patient's Gal index rates of change (Gal indexs RR) is classified rank test result with curative effect.
Table 3：RA patient's TNF-α inhibitor for treating result judgement.
Table 4：RA patient's Gal index rates of change (Gal indexs RR) is classified rank test result with curative effect.
Specific implementation method：
The present invention based on inventor above-mentioned field research, there is provided it is a kind of based in mammal (preferred people) blood The change of immunoglobulin G (IgG) surface double antenna complexity N sugar-chain end glycosyl galactose is thin to autoimmune disease patient The new method of intracellular cytokine targeted therapy sensitivity examination, state of illness monitoring and/or curative effect evaluation.The method of the present invention has filled up ability Blank of the domain in terms of sensitivity examination, with sensitivity it is high, specificity is good, simple operation characteristic, for precisely treatment, excellent Change government resources configuration, reduce sufferer burden and have great importance.
The present invention treats hyperreaction patient with Gal indexs in subsensitivety blood samples of patients not based on cytokine targeting preparation Together, high quick and hypoallergenic crowd is distinguished, the sensitivity examination to autoimmune disease patient's Cytokine Targeted Therapy is realized；Pin Before and after dependency and treatment to Gal indexs and autoimmune disease patient's coincident with severity degree of condition or disease different times The change of Gal indexs, realizes to autoimmune disease patient's state of illness monitoring and/or curative effect evaluation, and thus provides a kind of high Examination/the detection method of sensitive, high specific.
Inventor has found the sensitivity of Gal indexs of the invention and autoimmune disease Cytokine Targeted Therapy Property, the order of severity of disease and therapeutic effect height correlation.
Specifically, inventor has carried out following test and has drawn corresponding conclusion with the present invention：
1) the AS serum samples of 111 Jing TNF-α inhibitor for treating are detected, it was demonstrated that Gal indexs are exempted to itself The sensitivity examination of epidemic disease Disease Cytokine Targeted Therapy has high specific, susceptiveness and accuracy；
2) 111 AS clinical samples are carried out detecting follow-up visit monitoring research, finds the change of Gal indexs and the journey that is in a bad way Degree is related to curative effect, it was demonstrated that Gal indexs can be used for the state of an illness and curative effect monitoring.
3) the RA serum samples of 98 Jing TNF-α inhibitor for treating are detected, it was demonstrated that Gal indexs are to autoimmune The sensitivity examination of property Disease Cytokine Targeted Therapy has high specific, susceptiveness and accuracy；
4) 98 RA clinical samples are carried out detecting follow-up visit monitoring research, finds the change of Gal indexs and the journey that is in a bad way Degree is related to curative effect, it was demonstrated that Gal indexs can be used for the state of an illness and curative effect monitoring.
Result above confirms that Gal indexs can be independently used for the sensitivity sieve of autoimmune disease Cytokine Targeted Therapy Look into, and state of illness monitoring and/or curative effect evaluation are carried out to patient.
Relational language is defined
All numerical rangies provided herein be intended to clearly to include falling all numerical value between endpoints of ranges and it Between numerical range.The feature that the feature or embodiment that can be mentioned to the present invention is mentioned is combined.This specification is taken off The all features shown can be used in combination with any combinations thing form, and each feature disclosed in description can provide phase with any The alternative characteristics of same, impartial or similar purpose replace.Therefore except there is special instruction, disclosed feature is only impartial or similar The general example of feature.
As used herein, " contain ", " having " or " including " include "comprising", " mainly by ... constitute ", " substantially By ... constitute ", and " by ... constitute "；" mainly by ... constitute ", " substantially by ... constitute " and " by ... constitute " Belong to the subordinate concept of " containing ", " having " or " including ".
As used herein, term " Gal indexs (Gal Index, GI) " refers to object blood IgG surfaces double antenna complexity Galactose (Gal is removed in the terminal galactosylation quantitative change of N sugar chains, specially double antenna complexity N sugar-chain end0) sugared with galactose Chain (Gal2) abundance ratio, wherein, Gal0Corresponding to the double antenna complexity N- sugar chain that end connects without galactose, Gal2It is right The double antenna complexity N- sugar chain of 2 galactose should be connected with end.
As used herein, term " double antenna complexity N- sugar chain " is referred to：Connect on the core pentasaccharides having in N- sugar chains Enter the branched carbohydrate chains of 2 non-pure mannose, just like antenna-like (referring to looking into the good chief editor of stannum,《Biochemistry》People's Health Publisher, 2008 the 7th edition, the 454-455 page).
Exemplary Gal0And Gal2Structure it is as follows, wherein, GlcNAc represents N-Acetyl-D-glucosamine, and Man is represented Mannose, Gal represent galactose, and Fuc represents fucose.
As used herein, term " specificity ", " sensitivity ", " coincidence rate " are had with corresponding medical statisticses technics There is identical implication.Herein " specificity " (specificity) refer to clinical judgment be to Cytokine Targeted Therapy not Ratio of the Jing Indexs measures of the present invention for negative (i.e. insensitive) in sensitive case." sensitivity " (sensitivity) is referred to During clinical judgment is the case sensitive to Cytokine Targeted Therapy, ratio of the Jing Indexs measures of the present invention for positive (i.e. sensitive) Example." coincidence rate " (accuracy) refers to true positives sample size and true negative sample size sum accounts for the ratio of total sample size, reflection Indexs measure result of the present invention and experimenter are to the Cytokine Targeted Therapy whether degree that sensitive 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. marginal 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 " cytokine " targeted therapy " refer to for the cell in autoimmune disease object because Son carries out magnetic target therapy.The cytokine can for TNF-α, IL-1 β (interleukin-1 ' beta ') or IL-6 (interleukin- 6).Cytokine Targeted Therapy can be the various treatments for target cytokine, for example with targeting and suppress the cell because The antibody or antibody coupling matter of son suppresses to the cytokine.
As used herein, term " sensitivity to Cytokine Targeted Therapy " refers to autoimmune disease patient to thin The responsiveness of intracellular cytokine targeted therapy.When the Cytokine Targeted Therapy adopts antibody for therapeutic agent, the sensitivity Refer to the responsiveness of autoimmune disease patient's antagonist treatment.Accordingly, " to Cytokine Targeted Therapy (as antibody is controlled Treat) hyperreaction patient " refer to there is good response degree to Cytokine Targeted Therapy (such as Antybody therapy), so as to be derived from The patient of good therapeutic effect；Conversely, " to Cytokine Targeted Therapy (such as Antybody therapy) subsensitivety patient " is referred to cell Factor targeted therapy (such as Antybody therapy) treatment without response or only faint response, so as to fail to respond to any medical treatment or unsatisfactory curative effect trouble Person.
Blood (including serum, blood plasma and whole blood) IgG and its separation and purification
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 adopted to carry out the test of the present invention.Institute Blood sample is stated available from various mammalian objects, preferred people, monkey, Canis familiaris L., horse, cattle, sheep, pig, Mus, rabbit etc..Art technology Personnel are appreciated that due to the difference on composition, when the plasma sample and serum sample using experimenter is detected respectively When, Gal indexs may be slightly different.For purpose that is quantitative and comparing is easy to, serum sample is preferred in the present invention.
The method for separating IgG is known to persons of ordinary skill in the art, and methods described is included but is not limited to：IgG purification Post, salting out method, organic solvent precipitation method, Polyethylene Glycol displacement method, liquid chromatography (LC), affinity chromatography (such as protein A or Protein G parent Legal, polyamide composite film affinity method), as long as methods described does not destroy connected N- sugar chains on IgG.It is commercially available IgG Separating kit, such as purchased from Thermo Fisher Scientific.
Employ IgG purification columns in an embodiment of the invention to separate the IgG in sample, it is preferred to use albumen A purification columns, and using buffer solution and faintly acid drip washing buffer solution are combined with the alkalescence of purification column kit, it is more preferably described Purification column is high flux purification column, for example, can process the purification column of 96 samples simultaneously.
N sugar chains can be separated from IgG using method as known in the art, including but not limited to：Enzyme process, for example with sugar Glycosides enzyme, preferred glycosidase PNGase F；Chemical method, for example with glycoprotein hydrazinolysis reagent, such as ADM0155A hydrazinolysis test kit.
After N sugar chains are separated from IgG, N sugar chains can be separated and/or purification using method as known in the art, Methods described is included but is not limited to：Porous graphite carbon PGC solid phase extractions, polysaccharide/oligosaccharide purification column, agglutinin affinity method (such as continuous agglutinin affinity chromatography SLAC), capillary electrophoresis, efficiently/Ultra Performance Liquid Chromatography, organic reagent precipitation (such as 65% Ethanol) etc..
In an embodiment of the invention, porous graphite carbon PGC solid phase extractions are employed and separates N sugar chains, 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 adopt acetonitrile:Water:Trifluoracetic acid=25:74.95:(ratio is volume to 0.05 mixed solvent eluting sugar chain Than).
Galactose can be gone to the horizontal double antenna complexity N sugar-chain end in IgG surfaces using method as known in the art (Gal0), galactose sugar chain (Gal2) abundance be measured, as long as methods described can be carried out to glycosyl galactose level in N- sugar chains Quantitatively.Methods described is included but is not limited to：Mass spectrography, such as MALDI TOF MS method MALDI-MS, Fast atom bombardment mass spectroscopy FAB-MS, Electrospray Mass Spectrometry ESI-MS；Liquid chromatography；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip skill Art；Microflow control technique；A charge-coupled conjunction of nuclear magnetic resonance nmr or more mode.
Data analysing method
In the present invention, by Gal indexs, i.e. galactose is removed in the end of object blood IgG surfaces double antenna complexity N sugar chain (Gal0) and galactose sugar chain (Gal2) abundance ratio, as autoimmune disease Cytokine Targeted Therapy (as antibody is controlled Treat) sensitivity examination, the index of autoimmune disease state of illness monitoring and/or curative effect evaluation.
Gal index computing formula are as follows：
Gal indexs=aGal0/bGal2；Or
Can show that galactose (Gal is removed in the end of object blood IgG surfaces double antenna complexity N sugar chain0) sugared with galactose Chain (Gal2) abundance ratio any other formula, such as other variations such as its inverse, logarithm.
Wherein, between the respective value of a, b is separately located in more than 0 to 10, such as independently selected from 0.1,0.2,0.3, 0.4th, 0.5,0.6,0.7,0.8,0.9,1,1.2,1.5,2,2.5,3,3.5,4,5,6,7,8,9 or 10, wherein a ≠ 0, b ≠ 0). Can be according to various parameters Gal in the test instrunment and/or method for being adopted0And Gal2Responsiveness, the concrete value to a, b It is adjusted.For example, after being verified with standard method (such as Ultra Performance Liquid Chromatography, UPLC) based on the test method for being used Ratio, the concrete value of a, b is adjusted.
For example, the formula can be：Gal indexs (GI)=Gal0/Gal2, i.e. a=b=1.
The above-mentioned Gal for abundance ratio0And Gal2It is preferred that deriving from same sample, homogeneous detection and/or same spectrum Figure.Therefore, the deviation that the operating process such as the pretreatment of parallel sample can be avoided using the index to produce, so as to reduce Duplicate Samples This enters error during Mass Spectrometer Method, it is ensured that the high repeatability of analysis and accuracy.
Those of ordinary skill in the art can draw corresponding according to the detection data of different groups of objects based on common sense in the field Corresponding ROC curve is drawn according to the data of different crowd in ROC curve, such as embodiments 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, when those skilled in the art can easily find selection any threshold (i.e. cut-off values), the detection is right The identification ability of drug susceptibility, that is, the sensitivity screened, specificity and coincidence rate.
The calculating (calculating of threshold value) of screening results is depending 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, takes its corresponding Gal index for threshold value.
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).
With threshold value as boundary, it is judged to Gao Min higher than the value, is judged to hypoallergenic less than the value.
Preferred threshold range is 2.2-3.5 in the present invention, preferably 3.065.It will be understood by those skilled in the art that by In test population and the difference of concrete disease, the parameter meeting such as the selection of threshold value and its corresponding sensitivity, specificity and coincidence rate Difference, but be all contained in the threshold range of present invention restriction.Those skilled in the art can according to actually used demand, Requirement such as to specified disease, sensitivity, specificity etc., chooses appropriate from ROC curve disclosed by the invention or threshold range Preferred threshold value.Can be it is determined that after optimal threshold, the scope of optimal threshold ± 1～15% (be included any in the range of this Numerical point or subrange).
Also, also the disease progression and therapeutic effect of patient can be carried out according to the change of Gal indexs in the present invention Monitoring.As used herein, before and after " Gal index rates of change (Gal indexs RR) " refers to treatment and/or in intervals, Gal The intensity of variation of index.Gal index rate of change computing formula can be as follows：
Gal index RR=△ Gal indexs/Gal indexsBefore treatment× 100%
=(Gal indexsBefore treatment- Gal indexsAfter treatment)/Gal indexsBefore treatment× 100%
Wherein, the change of Gal indexs before and after △ Gal index expressions are treated, that is, represent subject disease interior for a period of time End of love；Or
Gal index RR=△ Gal indexs/Gal indexsBefore× 100%
=(Gal indexsBefore- Gal indexsAfterwards)/Gal indexsBefore× 100%
Wherein, the Gal indexs change between the untreated object different time points of △ Gal index expressions, that is, represent not Change of illness state of the Jing treatment targets within a period of time).
The change of Gal indexs can be divided into into multiple grades with monitoring disease according to the difference of Gal index RR intensity of variations Progress and/or curative effect.For example, can by Gal index rates of change be divided into RR 0 (rate of change≤5%), RR I (>5%～20%), RRⅡ(>20%～35%), RR III (>35%).Also dependent on needing and putting into practice, other classifications are carried out to the change of Gal indexs.
The application of Gal indexs and its rate of change
Gal indexs of the present invention and its rate of change can be applicable to carry out Cytokine Targeted Therapy to autoimmune disease In the purposes such as the sensitivity assessment of (such as TNF-α inhibitor for treating), state of illness monitoring and curative effect evaluation.These application including but not It is limited to：
(1) Gal indexs are applied to the sensitivity assessment of Cytokine Targeted Therapy treatment：When Gal indexs reach or high In threshold value set in advance, then judge that the experimenter carries out Cytokine Targeted Therapy and will obtain good therapeutic effect；Conversely, then judging The possibility for obtaining preferable curative effect is less.
(2) Gal indexs and its rate of change are used for into state of illness monitoring：To autoimmune disease sufferer treatment before and after or control Different time points during treatment or in disease process determine and calculate Gal indexs, when Gal indexs are higher than obtained by previous test Gal indexs are higher than scope set in advance (such as 5%～50% or the arbitrfary point in the range of this or subrange), then show this Sufferer disease progression or deterioration；Conversely, then sb.'s illness took a favorable turn.
(3) Gal indexs and its rate of change are used for into curative effect evaluation：Before and after Cytokine Targeted Therapy or in therapeutic process Different time points determine and calculate Gal indexs, when Gal indexs are higher than the Gal indexs obtained by previous test or higher than setting in advance (such as 5%～50% or the arbitrfary point in the range of this or subrange are (such as fixed scope>5%～20%)), then show the therapy And/or the unsatisfactory curative effect of medicine.
Advantages of the present invention
The present invention has following one or more advantages in the application：
(1) suitable for various autoimmune disease cells factor targeted therapy (such as Antybody therapy) sensitivity prediction and Examination；
(2) to the curative effect of various autoimmune disease and can be monitored；
(3) detect convenient, take short；For single sample, detected value is reproducible, good stability；High pass can also be carried out Amount detection.
The present invention meets the operation requirement that outstanding Testing index should meet, and shows that Gal indexs are thin in autoimmune disease The sensitivity examination of intracellular cytokine targeted therapy (such as Antybody therapy) medicine, the aspect such as state of an illness detection and curative effect evaluation have clinical answering Practical 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 limit the scope of the present invention.Those skilled in the art can make appropriate modification, change to the present invention, these modifications It is within the scope of the present invention with variation.
The experimental technique of unreceipted actual conditions in the following example, can be using the conventional method in this area or according to confession The condition proposed by business is answered, and also test can be provided by commercial company.
Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.Unless otherwise defined, it is all used in text Specialty and scientific words and same meaning familiar to one skilled in the art institute.Additionally, any similar to described content or equal Deng method and material all can be applicable in the inventive method.Preferable implementation described in text only presents a demonstration it with material With.
Embodiment 1.Gal index is used for the sensitivity examination of ankylosing spondylitises (AS) patient TNF-α inhibitor for treating
1.1 test objective
Judge that Gal indexs are used for the meaning of ankylosing spondylitises (AS) patient TNF-α inhibitor sensitiveness examination.
1.2 content of the test
111 patients with ankylosing spondylitis are carried out with Embrel treatment, the serum before collecting treatment and in therapeutic process, The change of detection wherein Gal indexs, with reference to evaluation of clinical, judges that Gal indexs are screened in TNF-α inhibitor for treating sensitivity In meaning.
1.3 case sample situations
What 1.3.1 serum sample correspondence case was followed enters a group principle
(1) age is 18～65 years old；
(2) meet the AS New York Classifications standard of 1984 and HLA-B27 is positive；
(3) in the disease activity phase, i.e. Bath AS disease activity index (Bath AS disease activity Index, BASDAI, ankylosing spondylitises function index) >=40 (Visual Analog test table [visual analogue scale, VAS] 0～100), rachialgia VAS >=4 (VAS 0～10) of scoring；
(4) without other any biological preparation treatments；
(5) screen in first 4 weeks and DMARD medicines (disease modifying antirheumatic drug) were not used Treatment；
(6) NSAID medicines are such as taken before being selected in, then the dosage of the NSAID medicines at least stablizes 4 weeks；Such as taking dose is little In or equivalent to the glucocorticoid of 10mg/ days prednisones, then dosage at least stablizes 4 weeks；
(7) naturopathy is such as currently in use during examination, then the therapeutic modality at least stablizes 2 weeks.
1.3.2 the exclusion standard that serum sample correspondence case is followed
(1) clinical and iconography points out spinal column complete tetanus；
(2) during the acute and chronic infection, when having a medical check-up, find there is urinary system, cardiopulmonary, oropharynx, five official ranks infection sign person； Now suffer from or had active tuberculosiss medical history, or have Close contacts with active tuberculosiss patient in the recent period；B-mode, hepatitis C or HIV sufferers；
(3) there are the important organs such as severe cardiac, liver, kidney and blood, hormonal system and multiple sclerosiss pathological changes and medical history person；
(4) once receive anti-TNF Drug therapys or in 3 months, participate in any clinical drug trial；
(5) malignant tumor patient or there is the Susceptible population of family history；
(6) inflammatory bowel, activeness anterior uveitises or psoriatic；
(7) female patient of gestation and age of sucking.
1.4 medicines and administering mode
Etanercept for Injection (Pfizer's Pharmaceutical), 25mg/, subcutaneous injection, 2 times a week (dosage is 25mg/ time).
1.5Gal Indexs measure
Collect sample before and after above-mentioned experimental subject TNF-α inhibitor for treating (after treatment the sample collection time for administration after 12 weeks, refer to 1.6.2), detect Gal indexs.Concrete detection method is as follows：
1.5.1 from IgG purification in the blood sample of examination object
Use with reference to Thermo Fisher Scientific Protein A Spin Plate for IgG Screening Family handbook is implemented, specific as follows：
1. purification column and buffer solution (with reference to buffer solution and drip washing buffer solution) are balanced 30 minutes at room temperature；
2. the lid of purification column bottom is removed, is placed it in wash plate, remove the lid at the top of purification column, at each 200 μ l combination buffers are added to balance in hole；
3. device 500g rotating speeds assembled above are centrifuged 2 minutes；Repeat step (2) and (3) are each 1～2 time；
4. mix 70 μ l serum and buffer solution is combined with 100 μ l, in adding the hole of purification column；Purification column is incubated under room temperature 30-60 minutes；
5. 500g centrifugal purifications post 2 minutes, collect sample solution (being placed in 96 orifice plates), repeat loading 1～2 time；
6. purification column is placed on washing/collecting board, in every hole, adds 400 μ l to combine buffer solution, 500g is centrifuged 2 points Clock, this step are repeated 3 times；
7. add 20 μ l to combine buffer solution in each hole of collecting board, purification column is placed on collecting board, and in each of which 200 μ l drip washing buffer solution are added in hole, is incubated 1 minute, be centrifuged 2 minutes with the rotating speed of 500g, repeat this step 2 time；Use BCA Sample is collected in test kit detection, determines the collection liquid residing for IgG, collects the IgG albumen being purified into；
8. 400 μ l drip washing buffer solution are added in each hole of purification column, wash 2 times, then with 400 μ l's 0.02% Hydrazoic acid,sodium salt is washed three times, makes purification column regeneration；
9. 100 μ l Hydrazoic acid,sodium salt are added in each hole of purification column or buffer solution is combined, is closed the lid, being put into can be close 4 DEG C of preservations in the sack of envelope.
1.5.2 the separation of sugar chain and purification
(A) by glucosides ferment treatment, dissociate from IgG sugar chain
1. in advance with 70% 96 orifice plate of washing with alcohol, then use milli-Q water；By mixing purification IgG sample solutions with Glycosidase (New England Biolabs companies, 0705s), and 96 orifice plates of above-mentioned mixed liquor addition (Corning, 3504), 37 DEG C The free sugar chain of reaction 12-24 hours.
(B) separation of sugar chain and purification
96 orifice plates of graphitiferous carbon are activated with the acetonitrile solution containing trifluoroacetic 80% (v/v) of 0.1% (v/v) Afterwards, each hole is balanced with 0.1% trifluoroacetic acid aqueous solution；Then by 96 orifice plates of enzymolysis sample and 96 orifice plates of graphitiferous carbon Assembling, 1000g centrifugation 2min；Deionized water wash impurity and salinity, finally with containing 0.05% (v/v) trifluoroacetic 25% (v/v) acetonitrile solution eluting sugar chain collect eluting solution.
1.5.3 the fluorescent labeling of sugar chain
1. by the sugar chain solution and standard solution (Gal of purification0/Gal2=1:1, m/m) lyophilizing or vacuum are outstanding dry；
2. 3 μ l are contained the DMSO/HAc of 50mg/ml 2- aminobenzamides (2-AB), 60mg/ml sodium cyanoborohydrides (7/3, v/v) solution is added in the sugar chain being dried obtained by step 1, while arranging blank；
3. step 2 gained sample and blank vortex centrifugal are incubated into 17-24 hours under the conditions of 37 DEG C；
4. add 30-50 μ l ultra-pure waters in step 4 gained sample cell, mix, 13000rpm, 4 DEG C of centrifugation 10-15 minutes, Transfer supernatant carries out ultra high efficiency liquid phase (UPLC) analysis in suitable sample bottle after labelling.
1.5.4 the ultra high efficiency liquid phase quantitative analyses of sugar chain
Instrument and parameter setting
Ultra Performance Liquid Chromatography instrument (Waters ACQUITY UPLC H-Class), chromatographic column (ACQUITY BEH Amide, 2.1 × 100mm, 1.7 μm, Waters), 100mM ammonium formate solution and acetonitrile of the mobile phase for pH 4.5, column temperature For 60 DEG C, it is M5 that flow velocity is 500 μ, 0 speed-1, the speed of λ 1 is M5rsmm, and λ λ speed is M5rsmm, and analysis time is 25.67min, liquid phase bar Part is gradient elution (0～25.67min, 78%～55.9% acetonitrile (pure solution).With 100mM ammonium formates as mobile phase A, pure second Nitrile is Mobile phase B, gradient elution, according to condition of gradient elution, by the ratio of two kinds of mobile phases of liquid phase the automatic control of pump.
1.5.5 the determination method of two types glycosyl galactose sugar chain chromatographic peak
In chromatogram, the sugar chain (Gal of different glycosyl galactoses0、Gal2) retention time it is different, by the spectrogram of sample with The oligosaccharide standards chart adding contrast of 2-AB labellings, determines Gal in sample chromatogram figure0、Gal2Retention time.
1.5.6 determination methods of glycosyl galactose degree
It is fixed examination ring polymer IgG surfaces double antenna complexity N sugar-chain end glycosyl galactose degree to be carried out using UPLC Amount com-parison and analysis, the double antenna complexity N sugar-chain end for calculating serum according to the size of peak height or peak area remove galactose with half Lactose sugar chain abundance ratio (Gal indexs, GI), and in this, as IgG surfaces glycosyl galactose quantitative change index.Institute in following examples With computing formula it is：
GI=[Gal0/Gal2)]
All of above Virus monitory and data processing are implemented under double-blind conditions.
1.6 parameters for observation on effect and result judgement
1.6.1 observation index
BASDAI, BASFI (ankylosing spondylitises function index), patient global evaluation (PGA), night backache and the overall back of the body Pain VAS scores.VAS standards of grading：It is 0 point, painless；1-3 point, mild pain；4-6 point, moderate pain；7-10 point, severe pain.
1.6.2TNF- alpha inhibitor treatment efficacy result judges
Through the treatment of 12 weeks, according to ankylosing spondylitises the standard of curative effect evaluation (ASAS) judge curative effect [Sengupta R, Stone MA.The assessment of ankylosing spondylitis in clinical practice.Nat Clin Pract Rheumatol,2007,3(9):496-503.]
According to parameters for observation on effect, comprehensive assessment TNF-α inhibitor curative effect.
The patient for being met following 2 conditions simultaneously is judged to TNF-α inhibitor for treating effective (ASAS20)：
(1) having 3 to obtain more than 20% below in 4 indexs improves, or improves at least 1 point of amplitude (VAS scoring 0-10)： ①BASFI；②PGA；3. hypnalgia and the scoring of rachialgia VAS；4. (Bath is tetanic, and type spinal column inflammatory Disease Activity refers to BASDAI Number) in the last two VAS average scores related to morning stiffness；
(2) another index before treatment compared with baseline without deterioration.
Conversely, remaining crowd is judged to invalid group of TNF-α inhibitor for treating.
All of above data processing is implemented under double-blind conditions.
The ASAS50 referred in 2.5 is referred to：
(1) having 3 to obtain more than 50% below in 4 indexs improves, or improves at least 2 points of amplitude (VAS scoring 0-10)： ①BASFI；②PGA；3. hypnalgia and the scoring of rachialgia VAS；4. (Bath is tetanic, and type spinal column inflammatory Disease Activity refers to BASDAI Number) in the last two VAS averages related to morning stiffness；
(2) another index before treatment compared with baseline without deterioration.
1.7 result
1.7.1AS patient's TNF-α inhibitor for treating result judgement
According to parameters for observation on effect ASAS20, AS patient 111, judges whether TNF-α inhibitor for treating is effective.Such as 1 institute of table Show, effective percentage is 72.97%.Accordingly, patient is divided into into TNF-α inhibitor for treating height quick group and TNF-α inhibitor for treating hypoallergenic Group, wherein high quick group of 81 people, 30 people of hypoallergenic group.Before two groups of patient, Gal desired value differences are extremely notable.
Table 1：AS patient's TNF-α inhibitor for treating result judgement
1.7.2Gal differentiation of the index to quick group of AS patient's TNF-α inhibitor height and TNF-α inhibitor hypoallergenic group
Before analysis treatment, Gal indexs are to quick group of TNF-α inhibitor height and the differentiation situation of TNF-α inhibitor hypoallergenic group：Receive The analysis result of examination person's performance curve (ROC) shows that Gal indexs are one and distinguish AS patient's TNF-α inhibitor well The Testing index of sensitivity.Data results show, area under curve (AUC) value of Gal indexs be 0.9418 (95%CI, 0.9004 to 0.9831).With reference to the criterion (as follows) of AUC, this index is used as diagnosis index pin-point accuracy.
AUC (area-under-the-curve) criterion (Clin.Chem.2007,53：1615-22.)：ROC is bent Line is usually used in identification ability of the multilevel iudge diagnosis index to disease in clinic.In theory, when AUC >=0.9, the diagnosis refers to Mark is considered as " pin-point accuracy "；When AUC is between 0.7≤AUC<0.9 and 0.5<AUC<When 0.7, respectively " it is suitable for " and " nothing Effect ".
In the case where cutoff value is set to 3.065, Gal indexs can distinguish well TNF-α inhibitor it is high quick group and TNF-α inhibitor hypoallergenic group, susceptiveness have reached 90%, and specificity is then 87.65% (referring to Fig. 1).
Result above shows that Gal indexs can be good at distinguishing TNF-α inhibitor high quick group and TNF-α suppression before the treatment Preparation hypoallergenic group, points out the index to can be used for the sensitivity examination of AS patient's TNF-α inhibitor for treating.
Embodiment 2.Gal index is used for the curative effect monitoring of patients with ankylosing spondylitis TNF-α inhibitor for treating
2.1 test objective
Judge Gal indexs in state of illness monitoring and curative effect evaluation after patients with ankylosing spondylitis TNF-α inhibitor for treating Meaning.
2.2 content of the test
Collect to the serum before 111 patients with ankylosing spondylitis Embrel treatments and in therapeutic process, detection is wherein The change of Gal indexs, the change of illness state situation joint assessment with patient judge that Gal indexs are monitored and curative effect in AS conditions of patients Meaning in assessment.
2.3 sample
With embodiment 1.
2.4Gal Indexs measure
2.4.1Gal index
With embodiment 1.
2.4.2Gal index rate of change (Gal indexs RR) computational methods
Gal index RR=△ Gal indexs/Gal indexsBefore treatment× 100%
=(Gal indexsBefore treatment- Gal indexsAfter treatment)/Gal indexsBefore treatment× 100%.
Wherein, the change of Gal indexs before and after △ Gal index expressions are treated.
According to the difference of Gal index RR intensity of variations, the change of Gal indexs is divided into into RR 0 (rate of change≤5%), Gal Index RR I (>5%～20%), Gal index RR II (>20%～35%), Gal index RR III (>35%).
2.5 efficacy determination
<ASAS20 (shows without obvious curative effects) that ASAS20 (shows effectively) that ASAS50 (shows evident in efficacy).
2.6 statistical method
Completely randomized design variance analyses are combined using rank test.First according to inspection sum of ranks result, it was demonstrated that per group it Between whether have significant difference；Simultaneously according to rank test rank average, visual evaluation each group curative effect.
2.7 result
Table 2.AS patient Gal indexs RR and curative effect relevance evaluation
Chi-square, 62.987；Df, 3；p<0.01
As a result show, with increasing for Gal index RR ranks, patient without obvious curative effects (<ASAS20)、ASAS20、 Distribution in ASAS50 gradually increases, and rank test confirms per there is significant difference between group.As shown in table 2, mean rank order Value (Mean Rank) gradually increases as Gal index RR ranks increase.On the one hand result above shows the change energy of Gal indexs Enough reflect the alleviation degree of disease, on the other hand also indicate that the index can be used in the curative effect evaluation of AS.
Embodiment 3.Gal index is used for rheumatoid arthritiss (RA) patient TNF-α inhibitor for treating sensitivity examination
3.1 test objective
Judge that Gal indexs are used for the meaning of patient with rheumatoid arthritis TNF-α inhibitor sensitiveness examination.
3.2 content of the test
98 patient with rheumatoid arthritis are carried out with Embrel treatment, the blood before collecting treatment and in therapeutic process Clearly, the change of detection wherein Gal indexs, with reference to evaluation of clinical, judges Gal indexs in TNF-α inhibitor for treating sensitivity Meaning in screening.
3.3 case sample situations
What 3.3.1 serum sample correspondence case was followed includes principle
(1) age is 18～65 years old, male or female；
(2) American Rheumatism Association (ACR) the RA criteria for classifications formulated in 1987 are met；
(3) treat more than 6 months or more than 1 year through NSAID (non-steroidal anti-inflammatory drug) and two or more antirheumatic, activity at the state of an illness Phase, treat without any other biological preparation, unused glucocorticoid treatment in 4 weeks.
3.3.2 the exclusion standard that serum sample correspondence case is followed
(1) severe cardiac, liver, renal damage and blood, endocrinopathy, acute and chronic infection, the past active tuberculosiss；
(2) once receive anti-TNF Drug therapys or in 3 months, participate in any clinical drug trial；
(3) malignant tumor patient or there is the Susceptible population of family history；
(4) female patient of gestation and age of sucking.
3.4 medicines and administering mode
Etanercept for Injection (beneficial general match, in marine Xin Guojian pharmaceutcal corporation, Ltds, S20050058), 25mg/ bottles, skin Lower injection, 2 times a week (dosage is 25mg/ time).
3.5Gal Indexs measure
With 1.5 parts of embodiment 1.
3.6 parameters for observation on effect
According to American society of rheumatism the standard of curative effect evaluation (ACR) specified value ACR 20, the choosing of disease activity degree evaluation Use DAS28.ACR 20 requires that swelling and Tender Joint Count improve up to 20%, and at least 3 improvement in following parameter Reach 20%：1) total evaluation of patient；2) total evaluation of doctor；3) patient is to Pain scale evaluation；4) forfeiture of function Degree；5) ESR CRP level.
3.7 result
3.7.1RA patient's TNF-α inhibitor for treating result judgement
According to parameters for observation on effect, RA patient 98, judges whether TNF-α inhibitor for treating is effective.As shown in table 3, effectively Rate is 76.53%.Accordingly, patient is divided into into TNF-α inhibitor for treating height quick group and TNF-α inhibitor for treating hypoallergenic group, wherein High quick group of 75 people, 23 people of hypoallergenic group.Two groups of Gal desired value differences are extremely notable.
Table 3：RA patient's TNF-α inhibitor for treating result judgement
3.7.2Gal differentiation of the index to quick group of RA patient's TNF-α inhibitor height and TNF-α inhibitor hypoallergenic group
Before analysis treatment, Gal indexs are to quick group of TNF-α inhibitor height and the differentiation situation of TNF-α inhibitor hypoallergenic group：Receive The analysis result of examination person's performance curve (ROC) shows that Gal indexs are one and distinguish RA patient's TNF-α inhibitor well The index of sensitivity.Data results show that area under curve (AUC) value of Gal indexs is 0.9441.Arrange in cutoff value In the case of 3.065, Gal indexs can distinguish TNF-α inhibitor height quick group and TNF-α inhibitor hypoallergenic group, spirit well Quick property has reached 86.96%, and specificity is then 93.33% (referring to Fig. 2).
As a result show, Gal indexs can be good at distinguishing RA patient's TNF-α inhibitor height quick group and TNF-α before the treatment Inhibitor hypoallergenic group, points out the index to can be used for the sensitivity examination of RA patient's TNF-α inhibitor for treating.
Embodiment 4.Gal index is used for the curative effect monitoring of patient with rheumatoid arthritis TNF-α inhibitor for treating
4.1 test objective
Judge Gal indexs in the state of illness monitoring and curative effect evaluation of patient with rheumatoid arthritis TNF-α inhibitor for treating Meaning.
4.2 content of the test
98 patient with rheumatoid arthritis are carried out with Embrel treatment, the blood before collecting treatment and in therapeutic process Clearly, the change of detection wherein Gal indexs, clinical assessment curative effect, judge the dependency of the change of Gal indexs and clinical efficacy.
4.3 sample
With embodiment 3.
4.4 medicines and administering mode
With embodiment 3
4.5Gal Indexs measure
1) Gal index calculating methods：With embodiment 1；
2) Gal indexs RR (Gal index rates of change) are calculated and stage division：With embodiment 2.
4.6 curative effects quantify grade scale
With reference to American society of rheumatism the standard of curative effect evaluation (ACR) in 1987, after being treated to patient according to ACR20, ACR50 State is classified (Arnett FC, Edworthy SM, Bloch DA etc., The American Rheumatism Association 1987revised criteria for the classification of rheumatoid arthritis[J].Arthritis Rheum,1988:31(3):315-324)。
4.7 statistical method
Using rank test (Kruskal-Wallis Test).First according to rank test result, it was demonstrated that per between group be It is no to have significant difference；Simultaneously according to rank test rank average, visual evaluation each group curative effect.
Table 4.RA patient Gal indexs RR and curative effect relevance evaluation
Chi-square, 52.464；Df, 3；p<0.01.
As a result show, with increasing for Gal index RR ranks, patient without obvious curative effects (<ACR20)、ACR20、ACR50 In distribution gradually increase, rank test confirms per there is significant difference between group.As shown in table 4, mean rank order value (Mean Rank) gradually increase as Gal index RR ranks increase.On the one hand result above shows that the change of Gal indexs can react disease The alleviation degree of disease, on the other hand also indicates that the index can be used in the curative effect evaluation of RA.
The all documents referred in the present invention are all incorporated as reference in this application, 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 teachings for having read the present invention, those skilled in the art can To make various changes or modifications to the present invention, these equivalent form of values equally fall within the model limited by the application appended claims Enclose.

Claims (10)

1. a kind of Cytokine Targeted Therapy sensitivity prediction for autoimmune disease and screening, to autoimmunity Disease carries out the product of state of illness monitoring and/or curative effect evaluation, the product can for such as test kit, equipment, operable system and/ Or combinations thereof, the product includes：
(A) for determine the reagent of the terminal galactosylation degree of blood IgG surfaces double antenna complexity N sugar chain, instrument and/ Or system, which is used to determine double antenna complexity N- sugar chain abundance Gal that IgG face extremities connect without galactose0Connect with end There is double antenna complexity N- sugar chain abundance Gal of 2 galactose2；
For example, the reagent of one or more method for being selected from the group, instrument and/or system：Matrix Assisted Laser Desorption flies Row time mass spectrum method MALDI-MS, fast atom bombardment mass spectroscopy FAB-MS, Electrospray Mass Spectrometry ES-MS；Liquid chromatography；Ultra high efficiency liquid Phase chromatography；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip technology；Microflow control technique；Nuclear magnetic resonance nmr, preferred ultra high efficiency liquid phase Chromatography；
(B) it is optional, for calculating Gal0And Gal2Abundance ratio Gal indexs module and/or processor；
(C) it is optional, for judging whether object is sensitive to Cytokine Targeted Therapy, carrying out disease to object according to Gal indexs Feelings monitoring and/or the module and/or processor of curative effect evaluation；
In some embodiments, the blood is selected from：Serum, blood plasma and whole blood, more preferably serum；
In some embodiments, the Cytokine Targeted Therapy for TNF-α, IL-1 β (interleukin-1 ' beta ') and/or IL-6 (interleukin-6)；
In some embodiments, the Cytokine Targeted Therapy is Antybody therapy.
2. product as claimed in claim 1, it is characterised in that the product also includes one or more for being selected from the group：
A) for gathering and/or deal with objects the reagent and/or instrument of blood sample；
B) for separation and/or the reagent and/or instrument of purified blood serum and/or blood plasma；
C) for separation and/or the reagent and/or instrument of purified blood serum/blood plasma IgG；
D) for separating, the reagent and/or instrument of purification and/or enrichment 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 phase extractions (preferably using acetonitrile:Water:Trifluoracetic acid=80:19.9:0.1 activation PGC, using water:Trifluoracetic acid=99.9:0.1 balance PGC, using acetonitrile:Water:Trifluoracetic acid=25:74.95:0.05 mixed solvent eluting sugar chain), it is polysaccharide/oligosaccharide purification column, solidifying Plain affinity method (such as continuous agglutinin affinity chromatography SLAC) of collection, capillary electrophoresis, efficiently/Ultra Performance Liquid Chromatography, organic reagent Precipitation is (such as 65% ethanol)；
E) for the reagent and/or instrument of labelling (such as fluorescent labeling) IgG surfaces N- sugar chains；
F) the double antenna complexity N sugar-chain end for storing and/or dealing with objects serum goes galactose rich with galactose sugar chain Degree ratio is used as the data base of IgG surfaces glycosyl galactose quantitative change index, module and/or processor；
G) for providing the module and/or processor of judgment threshold；
H) for judging that object is whether sensitive to Cytokine Targeted Therapy, carry out pre- state of illness monitoring to object according to Gal indexs And/or the module and/or processor of curative effect evaluation；
I) for offer diagnosis and/or the module and/or processor of testing result and/or report；
J) description or guide for use, wherein describing following one or more application and/or judgment mode：
I () Gal indexs are applied to the sensitivity assessment of Cytokine Targeted Therapy：Set when Gal indexs are reached at or above in advance Fixed threshold value, then judge that the experimenter is sensitive to Cytokine Targeted Therapy or is suitable for use with Cytokine Targeted Therapy；Conversely, Then judge that the experimenter is insensitive to Cytokine Targeted Therapy or be unsuitable for adopting Cytokine Targeted Therapy；
(ii) Gal indexs are changed for state of illness monitoring：To autoimmune disease sufferer treatment before and after, in therapeutic process or The different time points in ill each period determine and calculate Gal indexs, when Gal indexs higher than the Gal indexs obtained by previous test or Higher than scope set in advance (such as 5%～50% or the arbitrfary point in the range of this or subrange), then show the sufferer state of an illness Progress deteriorates；
(iii) Gal indexs are changed for curative effect evaluation：During different before and after Cytokine Targeted Therapy or in therapeutic process Between point determine and calculate Gal indexs, when Gal indexs are higher than the Gal indexs obtained by previous test or be higher than scope set in advance (such as 5%～50% or the arbitrfary point in the range of this or subrange), then show the unsatisfactory curative effect of the therapy and/or medicine.
It is preferred that, (i) in threshold value be 2.2～3.5, more preferably 3.065；
It is preferred that, optimal threshold can be determined based on the value of maximum slope point or maximum sensitivity and specificity in ROC curve, and Preferred threshold range can be the scope of optimal threshold ± 1～15% (including any number point or subrange in the range of this).
3. product as claimed in claim 1 or 2, it is characterised in that the Gal0And Gal2Abundance ratio Gal indexs meter Calculating is carried out using the formula being selected from the group：
Gal indexs=aGal0/bGal2, or other variations such as its inverse, logarithm；Wherein, a, b independently are more than 0 to 10 In the range of numerical value, such as a and b independently selected from 1,2,3,4,5,6,7,8,9 or 10 number；
For example, the formula being selected from the group can be adopted to carry out ratio calculation：
Gal indexs=Gal0/Gal2；
Gal indexs=Gal0/2Gal2；Or
Other variations such as its inverse, logarithm.
4. product as claimed in claim 1, it is characterised in that the autoimmune disease is selected from：Ankylosing spondylitises, class Rheumatic arthritis, arthritic psoriasises, adolescent arthritis, plaque psoriasis, ulcerative colitiss, Crohn ' s disease, Systemic lupus erythematosus (sle), multiple sclerosiss, systemic sclerosises, sjogren syndrome.
5. product as claimed in claim 1, it is characterised in that the product is used in the detection method for comprising the steps of：
(A') in measure object blood sample IgG surfaces double antenna complexity N sugar chain terminal galactosylation level, the end End Galactosylation levels include：The horizontal Gal of double antenna complexity N- sugar chain that IgG face extremities connect without galactose0And end End is connected with the horizontal Gal of double antenna complexity N- sugar chain of 2 galactose2；
(B') calculate Gal0And Gal2Abundance ratio, i.e. Gal indexs；
(C') judge whether object is sensitive to Cytokine Targeted Therapy according to Gal indexs, according to before and after treatment, in therapeutic process Or the Gal indexs change in ill each period is monitored to the state of an illness and/or curative effect is estimated.
6. product as claimed in claim 5, it is characterised in that methods described also includes one or more steps being selected from the group Suddenly：
(a') gather and/or deal with objects blood sample；
(b') separate and/or purified blood serum and/or blood plasma；
(c') separate and/or purified blood serum/blood plasma IgG；
(d') separation, purification and/or enrichment serum IgG surface N- sugar chains；
(e') labelling (such as fluorescent labeling) IgG surfaces N- sugar chains；
(f') the double antenna complexity N sugar-chain end for storing and/or dealing with objects serum goes galactose and galactose sugar chain abundance Ratio is used as IgG surfaces glycosyl galactose quantitative change index；
(g') provide judgment threshold；
(h') the Gal indexs of object are compared with threshold value to judge that object is whether sensitive to Cytokine Targeted Therapy, and is compareed Value is relatively monitored to the state of an illness and/or curative effect is estimated.
(i') testing result and/or report are provided.
7. product as claimed in claim 1, the product also include for judge treatment of autoimmune diseases effect its His test kit, equipment, system and/or combinations thereof.
8. whether reagent, instrument, module and/or processor are being prepared for judging the object with autoimmune disease to thin Intracellular cytokine targeted therapy is sensitive, the product of state of illness monitoring and/or curative effect evaluation is carried out to the object with autoimmune disease In application, the product can be such as test kit, equipment, system and/or combinations thereof, the reagent, instrument, module And/or processor includes：
(A) for determining the reagent of the terminal galactosylation level of blood IgG surfaces double antenna complexity N sugar chain, instrument, mould Block and/or processor, which is used to determine the horizontal Gal of double antenna complexity N- sugar chain that IgG face extremities connect without galactose0With End is connected with the horizontal Gal of double antenna complexity N- sugar chain of 2 galactose2；
For example, the reagent of one or more method for being selected from the group, instrument, module and/or processor：Ground substance assistant laser Parsing flight time mass spectrum MALDI methods, fast atom bombardment mass spectroscopy FAB-MS, Electrospray Mass Spectrometry ESI-MS；Liquid chromatography；Superelevation Effect liquid phase chromatogram；Liquid Chromatography-Mass Spectrometry；Carbohydrate chip technology；Microflow control technique；Nuclear magnetic resonance nmr；It is preferred that ultra high efficiency liquid Phase chromatograph；
(B) it is optional, for calculating Gal0And Gal2Abundance ratio Gal indexs module or processor；
(C) it is optional, for judging whether to carry out state of an illness prison to Cytokine Targeted Therapy sensitivity, to object according to Gal indexs Survey and/or the module or processor of curative effect evaluation,
In some embodiments, the Cytokine Targeted Therapy for TNF-α, IL-1 β (interleukin-1 ' beta ') and/or IL-6 (interleukin-6)；
In some embodiments, the Cytokine Targeted Therapy is Antybody therapy.
9. it is as claimed in claim 8 to apply, it is characterised in that the product is as any one of claim 1-7.
10. a kind of method screened for medicine and/or Therapeutic Method, methods described include：
(A ") measure object be administered and/or treat before, afterwards and/or during different time points blood sample in IgG tables The terminal galactosylation level of face double antenna complexity N sugar chain, the terminal galactosylation level include：IgG surfaces end Hold the horizontal Gal of double antenna complexity N- sugar chain without galactose connection0Double antenna complexity N- of 2 galactose is connected with end The horizontal Gal of sugar chain2；
(B ") calculates Gal0And Gal2Abundance ratio, i.e. Gal indexs；
(C ") judges the effectiveness of the medicine and/or Therapeutic Method according to Gal indexs or its change assessment, wherein when Gal refers to Absolute altitude in the 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 show the unsatisfactory curative effect of the medicine and/or Therapeutic Method.
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