CN110045125A - A kind of biomarker and application thereof for diagnosing retroperitoneal fibrosis - Google Patents
A kind of biomarker and application thereof for diagnosing retroperitoneal fibrosis Download PDFInfo
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Abstract
The invention discloses Rome snail lectins to prepare the purposes in the reagent for diagnosing retroperitoneal fibrosis.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 HPA agglutinin combination glycan is to reduce in IgG4-RD patient.Since HPA agglutinin is specific binding N- acetylgalactosamine, this shows that expression of the N- acetylgalactosamine level of glycosylation in IgG4-RD patient is to reduce.Further the IgG4-RD of three subgroups of research glycosylates expression, and expression quantity of the HPA agglutinin combination glycan levels in the patient of retroperitoneal fibrosis is minimum as the result is shown.Reduced HPA agglutinin combination glycan levels are used as the biological markers of diagnosis retroperitoneal fibrosis.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of biomarker for diagnosing retroperitoneal fibrosis and
Its purposes.
Background technique
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 provides a kind of for diagnosing the biomarker and its use of retroperitoneal fibrosis
On the way.
Firstly, the present invention provides a kind of for diagnosing the biomarker of retroperitoneal fibrosis, it is aggregated for Rome snail
Element is formed by compound in conjunction with IgG4.
Wherein, the IgG4 contains N- acetylgalactosamine.
Secondly, the use the present invention also provides the biomarker in the reagent for being used to prepare diagnosis retroperitoneal fibrosis
On the way.
Specifically, the diagnosis includes: that measurement is obtained from Rome in the biological sample that the diseases related patient of IgG4 is presented
Snail lectin is formed by the level of compound in conjunction with IgG4；Optionally,
Rome snail lectin is formed by compound in conjunction with IgG4 in the biological sample compared with contrasting data
It is horizontal, wherein relative to the contrasting data, Rome snail lectin is formed by compound in conjunction with IgG4 in the sample
Horizontal detectably reduce show a possibility that suffering from retroperitoneal fibrosis.
Wherein, the biological sample is blood serum sample.
Preferably, the level that Rome snail lectin is formed by compound in conjunction with IgG4 is surveyed by following steps
Amount, comprising:
Contact the biological sample from patient with Rome snail lectin；
B. agglutinin-glycan compound is formed between existing IgG4 and Rome snail lectin 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 Rome snails to coagulate
Collection element is preparing the purposes in the reagent for diagnosing retroperitoneal fibrosis.
Wherein, the Rome snail lectin 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
The form of the thin slice of 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.
On the other hand, the present invention also provides Rome snail lectins in preparing the reagent for diagnosing retroperitoneal fibrosis
Purposes.
Wherein, the diagnosis includes: and is obtained from Rome snail lectin and measurement the diseases related patient of IgG4 is presented
Biological sample contacted, measurement Rome snail lectin is formed by the level of compound in conjunction with IgG4；Optionally,
Rome snail lectin is formed by compound in conjunction with IgG4 in the biological sample compared with contrasting data
It is horizontal, wherein relative to the contrasting data, Rome snail lectin is formed by compound in conjunction with IgG4 in the sample
Horizontal detectably reduce show a possibility that suffering from retroperitoneal fibrosis.
Wherein, the biological sample is blood serum sample.
Preferably, the level that Rome snail lectin is formed by compound in conjunction with IgG4 is surveyed by following steps
Amount, comprising:
Contact the biological sample from patient with Rome snail lectin；
B. agglutinin-glycan compound is formed between existing IgG4 and Rome snail lectin 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 Rome snail lectins to prepare the use in the reagent for diagnosing retroperitoneal fibrosis
On the way.
Wherein, the Rome snail lectin 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
The form of the thin slice of 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.
On the other hand, the present invention also provides one kind for detecting and/or can quantitatively be aggregated with Rome snail in biological sample
A kind of diagnostic kit for the IgG4 that element combines, comprising: solid phase surface carrier, wherein the Rome snail lectin deposition
Or it is fixed on solid phase surface carrier, wherein after Rome snail lectin is formed by compound as peritonaeum in conjunction with IgG4
The biomarker of fibrosis.
In preferred embodiment of the invention kind, the kit further includes labeled and to carrying out biological sample
Antibody is reactive detection antibody.
Preferably, the solid phase surface carrier is latex pearl, porous flat plate or film item, nanotubes, with two dimensional code
The form of thin slice 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 HPA agglutinin combination glycan be in IgG4-RD patient reduction.Due to
HPA agglutinin is specific binding N- acetylgalactosamine, this shows that N- acetylgalactosamine level of glycosylation is suffered from IgG4-RD
Expression in person is to reduce.Further the IgG4-RD of three subgroups of research glycosylates expression, as the result is shown HPA agglutinin knot
It is minimum to close expression quantity of the glycan levels in the patient of retroperitoneal fibrosis.
Result of study shows, IgG4-RD patient, especially retroperitoneal fibrosis patient, HPA agglutinin combination glycan levels
It is to reduce, HPA agglutinin combination glycan levels can be used as the biological markers of retroperitoneal fibrosis medical diagnosis on disease.
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 HPA agglutinin signal value and compares (* *: P < 0.01).
Fig. 4 show Micoud Ritz disease and retroperitoneal fibrosis patient's HPA agglutinin signal value compares and ROC curve figure.
Fig. 5 show autoimmune pancreatitis and retroperitoneal fibrosis patient's HPA agglutinin signal value compares and ROC is bent
Line chart.
The correlation of IgG4 concentration after Fig. 6 is shown before purification.
Fig. 7 show the related of HPA 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, 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 the diagnosis of corresponding disease
Standard.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.Meet
Aforementioned three kinds of rules any one of agglutinin S/N data be identified as sharing 6 kinds of agglutinins with significant difference
(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, Rome snail lectin (HPA) signal value are overall in IgG4-RD group
It is to reduce (Fig. 3), further analyzes the distribution discovery of IgG4-RD subgroup HPA agglutinin signal value, A3 group retroperitoneal fibrosis
Value of the group in three subgroups is minimum (data and ROC diagnosis curve are shown in Fig. 4-5).In view of the combination N- acetyl half of HPA specificity
Lactose amine (GalNAc) glycan, thus infers, IgG4-RD patient, especially retroperitoneal fibrosis patient, the N- of IgG4 in serum
Acetylgalactosamine level of glycosylation is compared compared with other groups, and decreasing trend is presented, can be used as diagnosis and the mirror of retroperitoneal fibrosis
The biological markers not diagnosed.
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, or sugar
The actual change of base has used the second agglutinin microarray and agglutinin trace Dotblot to be verified.Second agglutinin
It 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 4 Fast 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, it is sweet with 20 μ l0.1M after washing 2 times
IgG4 is eluted to vacuum tube by propylhomoserin.With Dotblot identify lipidated protein, with protein silver staining kit (Beyotiome,
Shanghai, China) protein concentration is measured, 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 HPA agglutinin, the IgG4 Microarray signals value of the signal value and purifying of 4 microarray of serum IgG is proportional (Fig. 7).This
Showing the glycan that the HPA agglutinin in IgG4-RD patients serum combines --- N- acetylgalactosamine 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.Correlation analysis is as the result is shown: IgG4-RD patient's HPA agglutinin combination glycan
Content does not have correlation (table 2) with lab index.
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 of embodiment 4 167 IgG-RD patient IgG4 glycosylations and organ involvement
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.IgG4-RD patient HPA agglutinin combination glycan levels and more pancreas, bile duct are involved retroperitoneal fibrosis
Related (table 3).It is indicated above that after the reduction of IgG4-RD patient's HPA agglutinin combination glycan and pancreas, bile duct are involved peritonaeum
Fibrosis is related.The involvement of these organs is all common clinical manifestation, our result in retroperitoneal fibrosis patient
Show that HPA has differences in patient of these organs whether there is or not involvement, and difference is statistically significant, this can be used as the index and examines
Disconnected retroperitoneal fibrosis provides evidence.
3 IgG4-RD patient organ of table involvement is compared with IgG4 glycosylates content
There was no significant difference by NS*.
Claims (10)
1. it is a kind of for diagnosing the biomarker of retroperitoneal fibrosis, it is formed in conjunction with IgG4 by Rome snail lectin
Compound.
2. purposes of the biomarker described in claim 1 in the reagent for being used to prepare diagnosis retroperitoneal fibrosis.
3. purposes as claimed in claim 2, which is characterized in that the diagnosis includes: that measurement is obtained from presentation IgG4 correlation disease
Rome snail lectin is formed by the level of compound in conjunction with IgG4 in the biological sample of the patient of disease；Optionally,
Rome snail lectin is formed by the level of compound in conjunction with IgG4 in the biological sample compared with contrasting data,
Wherein, relative to the contrasting data, Rome snail lectin is formed by the water of compound in conjunction with IgG4 in the sample
Flat detectably reduce shows a possibility that suffering from retroperitoneal fibrosis.
4. Rome snail lectin is preparing the purposes in the reagent for diagnosing retroperitoneal fibrosis.
5. purposes as claimed in claim 4, which is characterized in that the diagnosis includes: to obtain Rome snail lectin with measurement
It is contacted from the biological sample that the diseases related patient of IgG4 is presented, measures Rome snail lectin institute's shape in conjunction with IgG4
At compound level；Optionally,
Rome snail lectin is formed by the level of compound in conjunction with IgG4 in the biological sample compared with contrasting data,
Wherein, relative to the contrasting data, Rome snail lectin is formed by the water of compound in conjunction with IgG4 in the sample
Flat detectably reduce shows a possibility that suffering from retroperitoneal fibrosis.
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 Rome snail lectin is formed by compound in conjunction with IgG4
Level is measured by following steps, comprising:
Contact the biological sample from patient with Rome snail lectin；
B. agglutinin-glycan compound is formed between existing IgG4 and Rome snail lectin 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 Rome snail lectin deposition is fixed on solid phase surface carrier
On, it is preferable that the solid phase surface carrier is latex pearl, porous flat plate or film item, nanotubes, the thin slice with two dimensional code
Deng form, it is preferable that the detection antibody by be covalently attached to enzyme, the marker with fluorescent chemicals or metal or
Marker with chemiluminescence compound marks.
9. a kind of for detecting and/or can quantitatively be wrapped with the kit of the IgG4 in conjunction with the snail lectin of Rome in biological sample
It includes: a kind of solid phase surface carrier, wherein the Rome snail lectin deposition is fixed on solid phase surface carrier, wherein
Rome snail lectin is formed by biomarker of the compound as retroperitoneal fibrosis in conjunction with IgG4, it is preferable that institute
Kit is stated to further include labeled and be 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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