CN110045127A - A kind of biomarker and application thereof of the diseases related multiple organ involvement of IgG4 - Google Patents
A kind of biomarker and application thereof of the diseases related multiple organ involvement of IgG4 Download PDFInfo
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- CN110045127A CN110045127A CN201910264599.2A CN201910264599A CN110045127A CN 110045127 A CN110045127 A CN 110045127A CN 201910264599 A CN201910264599 A CN 201910264599A CN 110045127 A CN110045127 A CN 110045127A
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention discloses datura stramonium lectin element in preparation for diagnosing the purposes in the reagent that the diseases related multiple organ of IgG4 is involved.The present invention 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 DSL agglutinin combination glycan is to reduce in IgG4-RD patient.Since DSL agglutinin is specific binding N-Acetyl-D-glucosamine, this shows that expression of the N-Acetyl-D-glucosamine level of glycosylation in IgG4-RD patient is to reduce.Further study show that DSL agglutinin combination glycan levels are related to multiple organ involvement, such as pancreas, salivary gland, lachrymal gland, retroperitoneal fibrosis, DSL agglutinin combination glycan levels can be used as the biological markers of IgG4-RD multiple organ involvement.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of biological marker of the diseases related multiple organ involvement of IgG4
Object and application thereof.
Background technique
IgG4 diseases related (IgG4related 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 diseases related multiple organ involvement of IgG4 biomarker and
Its purposes.
Firstly, the present invention provides a kind of biomarker of diseases related multiple organ involvement of IgG4, it is solidifying for datura
Collection element is formed by compound in conjunction with IgG4.
Wherein, multiple organ involvement include pancreas, salivary gland, lachrymal gland, retroperitoneal fibrosis, aorta and main artery by
It is tired.
Secondly, the present invention also provides the biomarkers of the diseases related multiple organ involvement of the IgG4 to be used in preparation
Diagnose the purposes in the reagent of the diseases related multiple organ involvement of IgG4.
Wherein, the diagnosis includes: that measurement is obtained from datura in the biological sample that the diseases related patient of IgG4 is presented
Agglutinin is formed by the level of compound in conjunction with IgG4；Optionally,
Datura stramonium lectin element 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, datura stramonium lectin element is formed by the water of compound in conjunction with IgG4 in the sample
Flat detectably reduce shows to suffer from a possibility that diseases related multiple organ of IgG4 is involved.
Wherein, the biological sample is blood serum sample.
Wherein, the level that datura stramonium lectin element is formed by compound in conjunction with IgG4 is measured by following steps, is wrapped
It includes:
Contact the biological sample from patient with datura stramonium lectin element agglutinin；
B. agglutinin-glycan compound is formed between existing IgG4 and datura stramonium lectin element 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.
Wherein, the datura stramonium lectin element is deposited or is fixed on solid phase surface carrier, it is preferred that the solid phase surface
Carrier is the form of latex pearl, porous flat plate or film item, nanotubes, thin slice with two dimensional code etc..
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.
Wherein, multiple organ involvement include pancreas, salivary gland, lachrymal gland, retroperitoneal fibrosis, aorta and main artery by
It is tired.
The present invention also provides a kind of datura stramonium lectin element in preparation for diagnosing the diseases related multiple organ involvement of IgG4
Purposes in reagent.
Wherein, the diagnosis includes: that measurement is obtained from datura in the biological sample that the diseases related patient of IgG4 is presented
Agglutinin is formed by the level of compound in conjunction with IgG4；Optionally,
Datura stramonium lectin element 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, datura stramonium lectin element is formed by the water of compound in conjunction with IgG4 in the sample
Flat detectably reduce shows to suffer from a possibility that diseases related multiple organ of IgG4 is involved.
Wherein, the biological sample is blood serum sample.
Wherein, the level that datura stramonium lectin element is formed by compound in conjunction with IgG4 is measured by following steps, is wrapped
It includes:
Contact the biological sample from patient with datura stramonium lectin element agglutinin；
B. agglutinin-glycan compound is formed between existing IgG4 and datura stramonium lectin element 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.
Wherein, the datura stramonium lectin element is deposited or is fixed on solid phase surface carrier, it is preferred that the solid phase surface
Carrier is the form of latex pearl, porous flat plate or film item, nanotubes, thin slice with two dimensional code etc..
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.
Wherein, multiple organ involvement include pancreas, salivary gland, lachrymal gland, retroperitoneal fibrosis, aorta and main artery by
It is tired.
The present invention also provides it is a kind of for detect and/or quantitative biological sample in can be with the IgG4 in conjunction with datura stramonium lectin element
Diagnostic kit, comprising: a kind of solid phase surface carrier, wherein the described datura stramonium lectin element deposition is fixed on solid phase table
On the carrier of face, wherein datura stramonium lectin element is formed by the conduct diseases related multiple organ of IgG4 of compound in conjunction with IgG4
Involvement.
Wherein, the diagnostic kit further includes labeled and is reactivity to the antibody for carrying out biological sample
Detection antibody.
Wherein, 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..
This research detects IgG4-RD patients serum IgG4 molecular surface by using agglutinin microarray and agglutinin is special
Property combine glycan spectrum, the results show that the content of DSL agglutinin combination glycan be in IgG4-RD patient reduction.Due to
DSL agglutinin is specific binding N-Acetyl-D-glucosamine, this shows N-Acetyl-D-glucosamine level of glycosylation in IgG4-RD
Expression in patient is to reduce.Further study show that DSL agglutinin combination glycan levels are related to multiple organ involvement, such as
Pancreas, salivary gland, lachrymal gland, retroperitoneal fibrosis etc., DSL agglutinin combination glycan levels can be used as the involvement of IgG4-RD multiple organ
Biological markers.
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 show IgG4-RD group, DC group and HC group DSL agglutinin signal value and compares (* *: P < 0.01).
The correlation of IgG4 concentration after Fig. 4 is shown before purification.
Fig. 5 show the related of DSL 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: three groups of crowds for being included in this research include: IgG4-RD group (167 IgG4-RD patients), DC group
(130 AID disease controls), HC group (86 physical examination of healthy population).Wherein the diagnosis of IgG4-RD group and DC group meets corresponding disease
The diagnostic criteria of disease.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.S/N data are shown
IgG4-RD patient group relative to the IgG4 of DC group and HC group glycosylate change: (1) by detection with Con A agglutinin (ConA),
The increase of the combination of Morniga M agglutinin (black mulberry) (MNA-M) and hairy vetch agglutinin (mannose is special), table
The increase (all P < 0.00) of bright mannose；(2) reduction combined by detection with wing pod crowtoe agglutinin (LTL), shows
The reduction (P < 0.00) of fucose；(3) reduction combined by detection with Rome snail lectin (HPA) shows N- acetyl half
The reduction (p < 0.00) of lactose amine (GalNAc)；(4) reduction combined by detection with datura stramonium lectin plain (DSL), shows N-
The reduction (p < 0.00) of acetylglucosamine (GlcNac).Fig. 3 shows IgG4-RD group, DC group and HC group DSL agglutinin letter
Number value compares.
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 to be verified.The micro- battle array of second agglutinin
Column are 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.By 20 μ l mouse anti-igg, 4 antibody (SouthernBiotech, Birmingham, USA) conjugation to 20 μ l pearls
(NHS-activated SepharoseTM 4Fast Flow,GE healthcare Life Sciences, Pittsburgh,
USA), 0.1M Tris-HCl is added then to seal excessive position.4 antibody of mouse anti-igg is cleaned with acid solution and aqueous slkali
Pearl 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, with 20 μ l 0.1M
IgG4 is eluted to vacuum tube by glycine.Lipidated protein is identified with Dotblot, with protein silver staining kit
(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. 4) with 4 level of serum IgG.The results show that right
For DSL agglutinin, the IgG4 Microarray signals value of the signal value and purifying of 4 microarray of serum IgG is proportional (Fig. 5).This
Showing the glycan that the DSL agglutinin in IgG4-RD patients serum combines --- acetylglucosamine level of glycosylation is abnormal
's.
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.In the various Laboratory Characteristics found in IgG-RD patient, IgG4-RD patient
The content and serum complement C3 level of DSL agglutinin combination glycan are positively correlated (r=0.36, P < 0.01) (table 2).
The correlation of the IgG4 glycosylation and Laboratory Characteristic of 2 167 IgG-RD patients of table
NS: no difference of science of statistics
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 4.IgG4-RD patient DSL agglutinin combination glycan levels are simultaneously related to multiple organ involvement, including pancreas, saliva
Liquid gland, lachrymal gland, retroperitoneal fibrosis, aorta and main artery involvement.
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 of the diseases related multiple organ involvement of IgG4, for datura stramonium lectin element in conjunction with IgG4 institute's shape
At compound.
2. biomarker described in claim 1 is in preparation for diagnosing in the reagent that the diseases related multiple organ of IgG4 is involved
Purposes.
3. purposes as claimed in claim 2, which is characterized in that the diagnosis includes: that measurement is obtained from presentation IgG4 correlation disease
Datura stramonium lectin element is formed by the level of compound in conjunction with IgG4 in the biological sample of the patient of disease；Optionally,
Datura stramonium lectin element is formed by the level of compound in conjunction with IgG4 in the biological sample compared with contrasting data,
In, relative to the contrasting data, the level that datura stramonium lectin element is formed by compound in conjunction with IgG4 in the sample can
Detection ground, which reduces, shows to suffer from a possibility that diseases related multiple organ of IgG4 is involved.
4. datura stramonium lectin element is in preparation for diagnosing the purposes in the reagent that the diseases related multiple organ of IgG4 is involved.
5. purposes as claimed in claim 4, which is characterized in that the diagnosis includes: to be obtained from datura stramonium lectin element with measurement
The biological sample that the diseases related patient of IgG4 is presented contacts, and measurement datura stramonium lectin element is formed by conjunction with IgG4
The level of compound；Optionally,
Datura stramonium lectin element is formed by the level of compound in conjunction with IgG4 in the biological sample compared with contrasting data,
In, relative to the contrasting data, the level that datura stramonium lectin element is formed by compound in conjunction with IgG4 in the sample can
Detection ground, which reduces, shows to suffer from a possibility that diseases related multiple organ of IgG4 is involved.
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 datura stramonium lectin element is formed by the water of compound in conjunction with IgG4
It is flat to be measured by following steps, comprising:
Contact the biological sample from patient with datura stramonium lectin element agglutinin；
B. agglutinin-glycan compound is formed between existing IgG4 and datura stramonium lectin element 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 datura stramonium lectin element deposition is fixed on solid phase surface carrier
On, preferred solid phase surface carrier is the shape of latex pearl, porous flat plate or film item, nanotubes, thin slice with two dimensional code etc.
Formula.
9. purposes as claimed in claim 7, wherein the detection antibody is by being covalently attached to enzyme, having fluorescent chemicals
Metal marker or marker with chemiluminescence compound mark.
10. a kind of for detecting and/or can quantitatively be wrapped with the kit of the IgG4 in conjunction with datura stramonium lectin element in biological sample
It includes: a kind of solid phase surface carrier, wherein the datura stramonium lectin element deposition is fixed on solid phase surface carrier, wherein graceful
Top sieve agglutinin is formed by the biomarker as the diseases related multiple organ involvement of IgG4 of compound in conjunction with IgG4,
Preferably, the kit further includes labeled and is reactive detection antibody to the antibody for carrying out biological sample, excellent
Selection of land, the solid phase surface carrier are the shapes of latex pearl, porous flat plate or film item, nanotubes, thin slice with two dimensional code etc.
Formula.
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