CN110045126A - A kind of biomarker and application thereof for diagnosis of autoimmune pancreatitis - Google Patents
A kind of biomarker and application thereof for diagnosis of autoimmune pancreatitis Download PDFInfo
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- CN110045126A CN110045126A CN201910264499.XA CN201910264499A CN110045126A CN 110045126 A CN110045126 A CN 110045126A CN 201910264499 A CN201910264499 A CN 201910264499A CN 110045126 A CN110045126 A CN 110045126A
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Abstract
The invention discloses purposes of the wing pod crowtoe agglutinin in the reagent that preparation is used for diagnosis of autoimmune pancreatitis.This research is composed by using the glycan of agglutinin microarray detection IgG4-RD patients serum IgG4 molecular surface and agglutinin specific binding, the results show that the content of LTL agglutinin combination glycan is to reduce in IgG4-RD patient.Since LTL agglutinin is specific binding fucose, this shows that expression of the fucose level of glycosylation in IgG4-RD patient is to reduce.Further the IgG4-RD of three subgroups of research glycosylates expression, expression quantity of the LTL agglutinin combination glycan levels in the patient of autoimmune pancreatitis is minimum as the result is shown, shows that LTL agglutinin combination glycan levels can be used as the biological markers of autoimmune pancreatitis medical diagnosis on disease.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of biological marker for diagnosis of autoimmune pancreatitis
Object and application thereof.
Background technique
Glycosylation is to modify one of most common form after protein transcription.Glycosylation can influence protein-protein interaction,
Cell-ECM identification, sticks and chemotaxis.More and more evidences show glycosylated change and disease under morbid state
Occurrence and development it is related, such as tumour, autoimmune disease.Therefore, disease can be improved in the glycosylated type of analyzing glucoprotein
The specificity of diagnosis
IgG4 diseases related (IgG4 related disease, IgG4RD) is new immune of one kind for recognizing in recent years
The auto-inflammatory disease of mediation.The disease with afflicted organ or hyperblastosis, enlargement, serum IgG 4 is horizontal significantly to be increased (>
1350mg/L), IgG4 positive lymphocyte infiltration (total 50% or more the thick liquid cell of IgG4 positive thick liquid cell Zhan) is in affected tissue
Main feature.This disease can be involved multiple organs such as lachrymal gland, salivary gland, pancreas, retroperitoneal tissue, bile duct, lung, kidney, prostate
Or tissue, clinical manifestation be Micoud Ritz disease, autoimmune pancreatitis, retroperitoneal fibrosis, autoimmune cholangitis,
Matter pneumonia, periorbit inflammatory pseudotumor etc..Autoimmunity disease summary (Autoimmunity Reviews) magazine is with entitled within 2010
" a kind of new syndrome birth: IgG4 related disease clinic spectrum " announces that this new disease gains public acceptance.2012 in the world
The comprehensive diagnos standard for announcing the disease for the first time makes its diagnosis be standardized.The pathological manifestations of IgG4-RD feature are lymphocyte
Infiltration, storiform fibrosis and obliterating phlebitis.IgG4-RD is early diagnosed, early treatment can prevent serious organ
Damage, tissue fibrosis, in addition it is dead.
Glycoprotein concentration in human serum is about 40g/L, is the splendid source for finding human diseases biomarker.With
RNA and protein are different, and the synthesis of the glycan sticked on glycoprotein does not need template.Glycosylation is mistake affected by many factors
Journey, comprising: cell type and its state of activation；Environmental factor, such as the presence of metabolin can be used；The age of cell, because of portion
Divide glycan that may lose over time；Inflammatory mediator, such as cell factor and chemotactic factor (CF).All of these factors taken together is all
It may change in the environment of autoimmunity.For example, some autoimmune diseases have characteristic cell factor.These
Cell factor is to glycosidase, and the expression of sialidase and glycosyl transferase has an impact, and these enzymes can directly affect glycan
Synthesis.In theory, characteristic immune state can show in the glycosylation of seroglycoid.
In view of important function of the glycosylation in disease, pass through high-throughput glycosylation analytical technology --- the micro- battle array of agglutinin
Column carry out the glycosylated expression of screening IgG4-RD patients serum IgG4, to inquire into the clinical application valence glycosylated in IgG4-RD
Value.
Summary of the invention
To solve the above-mentioned problems, the present invention provide a kind of biomarker for diagnosis of autoimmune pancreatitis and
Its purposes.
Firstly, the present invention provides a kind of biomarker for diagnosis of autoimmune pancreatitis, it is hundred arteries and veins of wing pod
Root agglutinin is formed by compound in conjunction with IgG4.
Wherein, wherein the IgG4 is containing fucose glycan.
Secondly, the use the present invention also provides the marker in the reagent for being used to prepare diagnosis of autoimmune pancreatitis
On the way.
Specifically, the diagnosis includes: that measurement is obtained from wing pod in the biological sample that the diseases related patient of IgG4 is presented
Crowtoe agglutinin is formed by the level of compound in conjunction with IgG4；Optionally,
Wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4 in the biological sample compared with contrasting data
Level, wherein relative to the contrasting data, wing pod crowtoe agglutinin is formed by multiple in conjunction with IgG4 in the sample
Horizontal detectably reduce for closing object shows a possibility that suffering from autoimmune pancreatitis.
Wherein, the biological sample is blood serum sample.
Preferably, the level that wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4 is surveyed by following steps
Amount, comprising:
Contact the biological sample from patient with wing pod crowtoe agglutinin；
B. agglutinin-glycan compound is formed between existing IgG4 and wing pod crowtoe agglutinin in the biological sample；
C. washing is to remove any unbonded IgG4；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. washing is to remove any unbonded labeled detection antibody；With
F. detectable signal is converted by the marker of the detection antibody.Secondly, the present invention also provides wing pod crowtoes
Purposes of the agglutinin in the reagent that preparation is used for diagnosis of autoimmune pancreatitis.
Wherein, the wing pod crowtoe agglutinin is deposited or is fixed on solid phase surface carrier.
Wherein, the solid phase surface carrier is preferably latex pearl, porous flat plate or film item, nanotubes, band two dimension
Any forms such as the thin slice of code.
Wherein, the detection antibody is by being covalently attached to enzyme, the marker with fluorescent chemicals or metal or having
The marker of chemiluminescence compound marks.
On the other hand, the present invention also provides wing pod crowtoe agglutinins is used for diagnosis of autoimmune pancreatitis in preparation
Purposes in reagent.
Wherein, the diagnosis includes: and is obtained from wing pod crowtoe agglutinin and measurement the diseases related trouble of IgG4 is presented
The biological sample of person contacts, and measurement wing pod crowtoe agglutinin is formed by the level of compound in conjunction with IgG4；Optionally
Ground,
Wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4 in the biological sample compared with contrasting data
Level, wherein relative to the contrasting data, wing pod crowtoe agglutinin is formed by multiple in conjunction with IgG4 in the sample
Horizontal detectably reduce for closing object shows to suffer from a possibility that diseases related multiple organ of IgG4 is involved.
Wherein, the biological sample is blood serum sample.
Preferably, the level that wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4 is surveyed by following steps
Amount, comprising:
Contact the biological sample from patient with wing pod crowtoe agglutinin；
B. agglutinin-glycan compound is formed between existing IgG4 and wing pod crowtoe agglutinin in the biological sample；
C. washing is to remove any unbonded IgG4；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. washing is to remove any unbonded labeled detection antibody；With
F. detectable signal is converted by the marker of the detection antibody.Secondly, the present invention also provides wing pod crowtoes
Purposes of the agglutinin in the reagent that preparation is used for diagnosis of autoimmune pancreatitis.
Wherein, the wing pod crowtoe agglutinin deposits or is fixed on solid phase surface carrier.
Wherein, the solid phase surface carrier is preferably latex pearl, porous flat plate or film item, nanotubes, band two dimension
Any forms such as the thin slice of code.
Wherein, the detection antibody is by being covalently attached to enzyme, the marker with fluorescent chemicals or metal or having
The marker of chemiluminescence compound marks.
On the other hand, the present invention also provides one kind for detecting and/or can quantitatively coagulate with wing pod crowtoe in biological sample
A kind of kit for the IgG4 that collection element combines, comprising: solid phase surface carrier, wherein the wing pod crowtoe agglutinin deposition
Or it is fixed on solid phase surface carrier, wherein wing pod crowtoe agglutinin is formed by compound as itself in conjunction with IgG4
The biomarker of immunity pancreatitis.
In a preferred embodiment of the present invention, the kit further includes labeled and to carrying out biological sample
Antibody is reactive detection antibody.
Preferably, wherein the solid phase surface carrier be preferably latex pearl, porous flat plate or film item, nanotubes,
Any form such as the thin slice with two dimensional code.
This research is by detecting IgG4-RD patients serum IgG4 molecular surface and agglutination by using agglutinin microarray
The glycan spectrum of element specific binding, the results show that the content of LTL agglutinin combination glycan is to reduce in IgG4-RD patient.
Since LTL agglutinin is specific binding fucose, this shows that expression of the fucose level of glycosylation in IgG4-RD patient is
It reduces.Further the IgG4-RD of three subgroups of research glycosylates expression, and LTL agglutinin combination glycan levels exist as the result is shown
Expression quantity in the patient of autoimmune pancreatitis is minimum.
Result of study shows, IgG4-RD patient, especially patients with autoimmune pancreatitis, LTL agglutinin combination glycan
Level is to reduce, and LTL agglutinin combination glycan levels can be used as the Biomarkers of autoimmune pancreatitis medical diagnosis on disease
Object.
Detailed description of the invention
Fig. 1 show 56 agglutinins (three wells) of the micro- permutation of agglutinin in the layout of array slides.
Fig. 2 show IgG4-RD patient's agglutinin microarray schematic diagram.
Fig. 3 IgG4-RD group, DC group and HC group LTL agglutinin signal value compare (* *: P < 0.01).
Fig. 4 show autoimmune pancreatitis and DC patient's LTL agglutinin signal value compares and ROC curve figure.
Fig. 5 show autoimmune pancreatitis and HC patient's LTL agglutinin signal value compares and ROC curve figure.
The correlation of IgG4 concentration after Fig. 6 is shown before purification.
Fig. 7 show the related of LTL agglutinin signal value between serum IgG 4 in agglutinin microarray and the IgG4 of purifying
Property.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Experimental specimen: be included in this research three groups of crowds include: IgG4-RD group (167 IgG4-RD patients, wherein
A1 group Micoud Ritz disease 59, A2 group autoimmune pancreatitis 50, A3 group retroperitoneal fibrosis 58), DC group (130
AID disease control), HC group (86 physical examination of healthy population).Wherein the diagnosis of IgG4-RD group and DC group meets corresponding disease
Diagnostic criteria.Enrolled crowd acquires new blood, and isolates serum immediately, -80 DEG C freeze it is spare.
The glycosylation of 1 agglutinin microarray analysis serum IgG 4 of embodiment
The agglutinin microarray being made of 56 kinds of agglutinin microchips.56 kinds of agglutinins are fixed to chip in triplicate
In, the total serum of each patient is subjected to 1:1000 dilution, is added in array, 4 DEG C of overnight incubations.Then, anti-igg 4-Cy3
Conjugation hybridizes with microchip 45 minutes in the dark.The fluorescence intensity of fluorescence intensity and low signal to all albumen has carried out solely
Vertical analysis.Chip image is converted into number format and is analyzed.
Each agglutination vegetarian refreshments is calculated using the signal-to-noise ratio (selecting moderate strength of the prospect relative to background) of each agglutination vegetarian refreshments
Signal-to-noise ratio (S/N).Biasing of the agglutinin microarray between array in order to prevent, we are normalized to normalizing using between array
Change S/N data.According to following rule, the significant difference of agglutinin combination vigor is determined by the data distribution in group: (1)
The agglutinin of IgG4-RD group is averaged, and (50% (IgG4-RD group) >=maximum value is (right by the maximum S/N that S/N is no less than in control group
According to group))；(2) the S/N lower quartile of IgG4-RD group is no less than upper quartile value (25% (the IgG4-RD group) of control group
>=75% (control group)；(3) the minimum S/N of IgG4-RD group be no less than control group intermediate value [minimum value (IgG4-RD group) >=
50% (control group)].
Using the agglutinin microarray containing 56 agglutinins come the glycosylation state (Fig. 1) in test experience sample.It is solidifying
The glycan molecule of the plain combination glycoprotein end that can be specific of collection, forms compound, passes through the spy of different agglutinin and glycan
The opposite sex is in conjunction with the type and content for carrying out research purpose protein surface glycan.Agglutinin microarray is because of its efficient feature, now
It is more and more widely used glycosylated research.After the sample equilibrium at room temperature frozen, it is added to agglutinin microarray, therewith instead
It answers, using washing, closing, the reaction of fluorescence secondary antibody and fluorescence detection, can get each agglutinin specific bond therewith
The signal value of glycan, signal value are related to binding affinity and bond strength (Fig. 2).
In order to ensure the fluorescence signal being collected into derives from the particular combination of IgG4, IgG4 antibody is marked using Cy3.Symbol
Close aforementioned three kinds of rules any one of agglutinin S/N data be identified as sharing 6 kinds of agglutinations with significant difference
Plain (table 1).
With the agglutinin of significant difference in 1 agglutinin microarray of table
The affinity signal value of 6 kinds of agglutinins, which is shown between three groups of samples, significant difference.Pass through agglutinin-glycan
Binding signal analysis, it has been found that comparison DC group and HC group, wing pod crowtoe agglutinin (Lotus tetragonolobus
Lectin) (LTL) signal value is generally (Fig. 3) reduced in IgG4-RD group, further analyzes IgG4-RD subgroup LTL agglutination
The distribution discovery of plain signal value, value of the A2 subgroup autoimmune pancreatitis in three subgroups minimum (data and ROC curve
Figure such as Fig. 4-5).In view of combination fucose (Fuc) glycan of LTL specificity, thus we infer, IgG4-RD patient, especially
It is patients with autoimmune pancreatitis, IgG4 fucosylation level is compared compared with other groups in serum, and decreasing trend is presented, can make
For the biological markers of diagnosis and the antidiastole of autoimmune pancreatitis.
The purification of 2 serum IgG 4 of embodiment and identification
In order to further determine IgG-RD patient it is glycosylated variation whether due to 4 concentration of serum IgG increase, still
Glycosylated actual change has used the second agglutinin microarray and agglutinin trace Dotblot to be verified.Second agglutination
Element is made of 6 kinds of agglutinins, including HPA, DSL, LTL, VVA mannose, MNA-M and ConA.Operation is the same.
IgG4 is isolated from serum by immuno-precipitation.Sample includes 12 IgG-RD patients, 3 DC patients and 1
Name HC patient.20 μ l mouse anti-igg, 4 antibody (SouthernBiotech, Birmingham, USA) is coupled to 20 μ l pearls
(NHS-activated SepharoseTM 4F ast Flow,GE healthcare Life Sciences,
Pittsburgh, USA), 0.1M Tris-HCl is added then to seal excessive position.It is cleaned with acid solution and aqueous slkali
4 antibody pearl of mouse anti-igg 3 times.Every pillar uses 5 μ l serum.It is to be incubated overnight by pillar.It is cleaned 8 times with PBST, after washing 2 times,
IgG4 is eluted to vacuum tube with 20 μ l 0.1M glycine.Lipidated protein is identified with Dotblot, with protein silver staining reagent
Box (Beyotiome, Shanghai, China) measurement protein concentration, all IgG4 samples are stored in -80 DEG C of progress subsequent processings.
By the comparison of IgG4 concentration and opposite 4 content of serum IgG to 16 patients after purification, find after purification
IgG4 concentration results have preferable correlation (r=0.593, P=0.015) (Fig. 6) with 4 level of serum IgG.The results show that right
For LTL agglutinin, the IgG4 Microarray signals value of the signal value and purifying of 4 microarray of serum IgG is proportional (Fig. 7).This
Show that LTL agglutinin in IgG4-RD patients serum combines glycan --- fucose level of glycosylation is abnormal.
The correlation analysis of the IgG4 glycosylation and Laboratory Characteristic of 3 167 IgG-RD patients of embodiment
Observe that there are significant differences between the agglutinin signal of IgG-RD patient, we further assess agglutinin signal
Relationship between clinical laboratory measures.Correlation analysis is as the result is shown: IgG4-RD patient's LTL agglutinin combination glycan
Content and serum IgA level are positively correlated (table 2).
The content and the correlation of Laboratory Characteristic of 2 167 IgG4-RD patient's agglutinin combination glycan of table
* NS: there was no significant difference
The relationship that 4 167 IgG-RD patient IgG4 glycosylations of embodiment are participated in organ
It is presented in various organ involvements in patient, the different level of agglutinin specific binding glycan is compared.
The results are shown in Table 3.It is indicated above that IgG4-RD patient LTL agglutinin combination glycan levels and more pancreas, bile duct are involved abdomen
Fibrosis correlation (table 3) after film.The results show that LTL combination glycan levels are in these organs of pancreas, bile duct and retroperitoneal fibrosis
Whether there is or not there are statistical differences in the patient of involvement, and the clinical characters of autoimmune pancreatitis also have the involvement of these organs
Performance.It can thus be the index diagnosis of autoimmune pancreatitis and evidence be provided.
3 IgG4-RD patient organ of table involvement is compared with IgG4 glycosylates content
There was no significant difference by NS*.
Claims (10)
1. a kind of biomarker for diagnosis of autoimmune pancreatitis is wing pod crowtoe agglutinin in conjunction with IgG4
It is formed by compound.
2. purposes of the biomarker described in claim 1 in the reagent for being used to prepare diagnosis of autoimmune pancreatitis.
3. purposes as claimed in claim 2, which is characterized in that the diagnosis includes: that measurement is obtained from presentation IgG4 correlation disease
Wing pod crowtoe agglutinin is formed by the level of compound in conjunction with IgG4 in the biological sample of the patient of disease；Optionally,
Wing pod crowtoe agglutinin is formed by the water of compound in conjunction with IgG4 in the biological sample compared with contrasting data
It is flat, wherein relative to the contrasting data, wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4 in the sample
Horizontal detectably reduce show a possibility that suffering from autoimmune pancreatitis.
4. purposes of the wing pod crowtoe agglutinin in the reagent that preparation is used for diagnosis of autoimmune pancreatitis.
5. purposes as claimed in claim 4, which is characterized in that the diagnosis includes: by wing pod crowtoe agglutinin and measurement
It is contacted obtained from the biological sample that the diseases related patient of IgG4 is presented, measures wing pod crowtoe agglutinin in conjunction with IgG4
It is formed by the level of compound；Optionally,
Wing pod crowtoe agglutinin is formed by the water of compound in conjunction with IgG4 in the biological sample compared with contrasting data
It is flat, wherein relative to the contrasting data, wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4 in the sample
Horizontal detectably reduce show a possibility that suffering from autoimmune pancreatitis.
6. purposes as claimed in claim 3 or 5, wherein the biological sample is blood serum sample.
7. purposes as claimed in claim 3 or 5, wherein wing pod crowtoe agglutinin is formed by compound in conjunction with IgG4
Level measured by following steps, comprising:
Contact the biological sample from patient with wing pod crowtoe agglutinin；
B. agglutinin-glycan compound is formed between existing IgG4 and wing pod crowtoe agglutinin in the biological sample；
C. washing is to remove any unbonded IgG4；
D. it adds being labeled and is reactive detection antibody to the antibody for carrying out biological sample；
E. washing is to remove any unbonded labeled detection antibody；With
F. detectable signal is converted by the marker of the detection antibody.
8. purposes as claimed in claim 7, wherein the wing pod crowtoe agglutinin deposition is fixed on solid phase surface load
On body, it is preferred that the solid phase surface carrier is latex pearl, porous flat plate or film item, nanotubes, with the thin of two dimensional code
The form of piece etc., it is preferred that the detection antibody by be covalently attached to enzyme, the marker with fluorescent chemicals or metal,
Or marker with chemiluminescence compound marks.
9. it is a kind of for detect and/or quantitative biological sample in can with the kit of the IgG4 in conjunction with wing pod crowtoe agglutinin,
It include: a kind of solid phase surface carrier, wherein the wing pod crowtoe agglutinin deposition is fixed on solid phase surface carrier,
Wherein, wing pod crowtoe agglutinin is formed by biological marker of the compound as autoimmune pancreatitis in conjunction with IgG4
Object, it is preferable that the kit further includes labeled and is reactive detection antibody to the antibody for carrying out biological sample.
10. kit as claimed in claim 9, which is characterized in that the solid phase surface carrier is latex pearl, porous flat plate
Or the form of film item, nanotubes, thin slice with two dimensional code etc..
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