CN110031632A - A kind of biomarker of Disease Activity and application thereof that reflection IgG4 is diseases related - Google Patents
A kind of biomarker of Disease Activity and application thereof that reflection IgG4 is diseases related Download PDFInfo
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- CN110031632A CN110031632A CN201910264593.5A CN201910264593A CN110031632A CN 110031632 A CN110031632 A CN 110031632A CN 201910264593 A CN201910264593 A CN 201910264593A CN 110031632 A CN110031632 A CN 110031632A
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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
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- 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
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- G01N2800/00—Detection or diagnosis of diseases
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Abstract
The invention discloses Morniga M agglutinins and/or hairy vetch agglutinin to prepare the purposes in the reagent for reflecting the diseases related Disease Activity of IgG4.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 glycan is raised in IgG4-RD patient in conjunction with MNA-M and VVA mannose agglutinin.Since MNA-M and VVA mannose agglutinin is the combination mannose of specificity, this shows that mannosylated level is that increasing is highly expressed in IgG4-RD patient.Further study show that mannosylated level is related to the activity of disease, and and exocrine gland, such as the involvement correlation of salivary gland and lachrymal gland.Hypermannosylation can reflect IgG4-RD patient disease activity, be used as the biomarker of IgG4-RD Disease Activity.
Description
Technical field
The invention belongs to field of biological detection, and in particular to a kind of life for the Disease Activity that reflection IgG4 is diseases related
Object marker and application thereof.
Background technique
Glycan is together with nucleic acid, protein, lipid, for four kinds of basic molecules for constituting life system.Glycoprotein surface connects
It connects, the glycan molecule with structure diversity is referred to as glycan.Glycosylation is that protein post-translational modification is most common and complex shape
One of formula influences the biological function of glycoprotein, such as maintains cell stability, adjusts cell activity, mediate cell adhesion
And secretion etc..When body is in inflammation and autoimmune state, the glycan sticked on glycoprotein can be with immune effector molecule
Interaction generates characteristic glycoforms, shows the glycosylation spectrum of disease specific.More and more evidences show
Characteristic glycosylation spectrum can be used as disease incidence mechanism, biomarker, other clinics and answer in autoimmune disease (AID)
With and disease control process index.
AID is one group of diseases associated with inflammation to generate autoantibody for body autoantigen, it includes more than 80 kinds diseases
Disease.IgG4 diseases related (IgG-RD) is one of AID.IgG4-RD was before 10 years by Japanese gastroenterologist
Doctor Kamisawa observes the identical pathology of AIP in other afflicted organs of autoimmune pancreatitis (AIP) patient
It being proposed when change, it is a kind of multiple organ fiber inflammatory disease, it is characterised in that the horizontal abnormality of patients serum IgG4 increases,
It forms inflammatory pseudotumor and generates the secreting type plasmablast of a large amount of IgG secretions 4 in the organ of involvement.Currently, for IgG4-
RD disease cognitive and research are at developing stage, while also lacking reliable index and disease work is diagnosed and reflected to disease
Dynamic property.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of biology mark of Disease Activity that reflection IgG4 is diseases related
Will object and application thereof.
Firstly, the present invention provides a kind of biomarker of Disease Activity that reflection IgG4 is diseases related, it is
Morniga M agglutinin and/or hairy vetch agglutinin are formed by compound in conjunction with IgG4.
Wherein, the IgG4 contains mannose glycan.
Secondly, the present invention also provides the marker in preparation for reflecting the diseases related Disease Activity of IgG4
Purposes in reagent.
Specifically, the reflection IgG4 diseases related Disease Activity includes: that measurement is obtained from presentation IgG4 correlation
Morniga M agglutinin and/or hairy vetch agglutinin are formed in conjunction with IgG4 in the biological sample of the patient of disease
Compound level；Optionally,
Compared with contrasting data in the biological sample Morniga M agglutinin and/or hairy vetch agglutinin with
IgG4 combines the level for being formed by compound, wherein relative to the contrasting data, Morniga M is aggregated in the sample
Horizontal detectably improve that element and/or hairy vetch agglutinin are formed by compound in conjunction with IgG4 can reflect IgG4
Diseases related Disease Activity.
Wherein, the biological sample is blood serum sample.
Preferably, Morniga M agglutinin and/or hairy vetch agglutinin are formed by compound in conjunction with IgG4
Level measured by following steps, comprising:
Contact the biological sample from patient with Morniga M agglutinin and/or hairy vetch agglutinin；
B. in the biological sample between existing IgG4 and Morniga M agglutinin and/or hairy vetch agglutinin
Form agglutinin-glycan compound；
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.
On the other hand, the present invention also provides Morniga M agglutinins and/or hairy vetch agglutinin is used in preparation
Reflect the purposes in the reagent of the diseases related Disease Activity of IgG4.
Wherein, the diagnosis includes: to be obtained from Morniga M agglutinin and/or hairy vetch agglutinin with measurement
The biological sample that the diseases related patient of IgG4 is presented contacts, and measures Morniga M agglutinin and/or long pubescence open country pea
Beans agglutinin is formed by the level of compound in conjunction with IgG4；Optionally,
Compared with contrasting data in the biological sample Morniga M agglutinin and/or hairy vetch agglutinin with
IgG4 combines the level for being formed by compound, wherein relative to the contrasting data, Morniga M is aggregated in the sample
Horizontal detectably improve that element and/or hairy vetch agglutinin are formed by compound in conjunction with IgG4 can reflect IgG4
Diseases related Disease Activity.
Wherein, the biological sample is blood serum sample.
Preferably, Morniga M agglutinin and/or hairy vetch agglutinin are formed by compound in conjunction with IgG4
Level measured by following steps, comprising:
Contact the biological sample from patient with Morniga M agglutinin and/or hairy vetch agglutinin；
B. in the biological sample between existing IgG4 and Morniga M agglutinin and/or hairy vetch agglutinin
Form agglutinin-glycan compound；
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 Morniga M agglutinin and/or hairy vetch agglutinin deposit or are fixed on solid phase surface
On carrier.
Wherein, the topical carrier is preferably latex pearl, porous flat plate or film item, nanotubes, with two dimensional code
The form of thin slice 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 Morniga M in biological sample
A kind of diagnostic kit for the IgG4 that element and/or hairy vetch agglutinin combine, comprising: solid phase surface carrier, wherein institute
The Morniga M agglutinin and/or hairy vetch agglutinin stated are deposited or are fixed on solid phase surface carrier, wherein
Morniga M agglutinin and/or hairy vetch agglutinin are formed by compound as reflection IgG4 phase in conjunction with IgG4
The biomarker of the Disease Activity of closing property disease.
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, the topical carrier is latex pearl, porous flat plate or film item, nanotubes, the thin slice with two dimensional code
Deng form.
The present invention 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 glycan is in IgG4-RD patient in conjunction with MNA-M and VVA mannose agglutinin
In be raised.Since MNA-M and VVA mannose agglutinin is the combination mannose of specificity, this shows mannose group
Change level is that increasing is highly expressed in IgG4-RD patient.Further study show that the mannosylated horizontal activity with disease
Correlation, and and exocrine gland, such as the involvement correlation of salivary gland, the parotid gland and lachrymal gland.
Human IgG contains a small amount of mannose structures as glycoprotein.Studies have shown that the IgG Fc with high mannose type is poly-
Sugar can enable IgG to be metabolized from serum faster by influencing IgG half-life period in conjunction with mannose receptor.Separately there is research to confirm,
The high mannose structures of IgG enhance antibody-dependant cell cytotoxic activity (ADCC) activity and affect IgG effector function.In addition,
The high-caliber mannosylated binding affinity reduced to C1Q, to weaken complement-dependent cytotoxicity (CDC)
Effect.In conjunction with ours as a result, we show that the prediction IgG4-RD disease that is used as of 4 hypermannosylation of serum IgG is lived
The potential marker of dynamic property.
IgG4-RD serum mannose base (MNA-M and VVA-mannose agglutinin reactivity) level is to increase expression
, hypermannosylation can reflect IgG4-RD patient disease activity, be used as the biology of IgG4-RD Disease Activity
Marker.
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 MNA-M agglutinin signal value compare (* *: P < 0.01).
Fig. 4 show IgG4-RD group, DC group and HC group VVA-mannose agglutinin signal value and compares (* *: P < 0.01).
The correlation of IgG4 concentration after Fig. 5 is shown before purification.
Fig. 6 show the phase of MNA-M agglutinin signal value between serum IgG 4 and the IgG4 of purifying in agglutinin microarray
Guan Xing
Fig. 7 show in agglutinin microarray VVA-mannose agglutinin signal between serum IgG 4 and the IgG4 of purifying
The correlation of value.
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.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 combines HC group, Morniga M agglutinin (black mulberry) (MNA-M) agglutinin signal value
It is increased (Fig. 3) in IgG4-RD group；Hairy vetch agglutinin (V.villosa, mannose are special) (VVA
Mannose) agglutinin signal value is increased (Fig. 4) in IgG-RD group.It is special in view of MNA-M and VVA mannose
The combination mannose residue of property, thus we infer, mannosylated level is compared compared with other groups in IgG4-RD patients serum,
The trend increased is presented.
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 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, 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. 5) with 4 level of serum IgG.The results show that right
For MNA-M and VVA mannose agglutinin, the signal value of 4 microarray of serum IgG and the IgG4 Microarray signals value of purifying
Proportional (Fig. 6-7).This shows that MNA-M the and VVA mannose agglutinin in IgG4-RD patients serum combines poly-
Sugar --- mannosylated level is abnormal.
The correlation point that the IgG4 glycosylation of 3 167 IgG-RD patients of embodiment is involved with Laboratory Characteristic and organ
Analysis
In order to further inquire into MNA-M and VVA mannose agglutinin combination glycan and clinical laboratory measures and target
The correlation of organ involvement, we analyze their correlation.Correlation analysis is as the result is shown: IgG4-RD patient MNA-M is solidifying
Collection element combines the content and change of serum C 3 of glycan, and C4 level is negatively correlated (table 2), and (table related to salivary gland and lachrymal gland involvement
3)；The content and serum CA125 of IgG4-RD patient's VVA mannose agglutinin combination glycan, C3, the negatively correlated (table of C4 level
2), and related (table 3) to salivary gland, lachrymal gland involvement.It is indicated above that the increase and disease of IgG4-RD patient's sweet dew sugar level
Activity it is related, and and exocrine gland, the involvement such as salivary gland and lachrymal gland it is related.
One of the characteristics of autoimmune disease is immunity disease, and immune indexes are disease extremely.When living in disease
When the dynamic phase, the amynologic index in patients serum can change, such as: c reactive protein, erythrocyte sedimentation rate increase, complement component C3, C4 drop
It is low, and after disease treatment is alleviated, these reaction Disease Activity indexs can then be restored to normal person's level accordingly.We
Result of study observe MNA-M and VVA mannose agglutinin combination IgG4 glycan be formed by complex levels and Complement C_3,
C4 level is negatively correlated, and P value, less than 0.05, thus deducibility is high-caliber mannosylated related to Disease Activity.
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
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 Disease Activity of reflection IgG4, be Morniga M agglutinin and/or
Hairy vetch agglutinin is formed by compound in conjunction with IgG4.
2. biomarker described in claim 1 is preparing the reagent for reflecting the diseases related Disease Activity of IgG4
In purposes.
3. purposes as claimed in claim 2, which is characterized in that the reflection IgG4 diseases related Disease Activity packet
Include: measurement is obtained from Morniga M agglutinin and/or long pubescence open country pea in the biological sample that the diseases related patient of IgG4 is presented
Beans agglutinin is formed by the level of compound in conjunction with IgG4；Optionally,
Morniga M agglutinin and/or hairy vetch agglutinin and IgG4 in the biological sample compared with contrasting data
In conjunction with the level for being formed by compound, wherein relative to the contrasting data, in the sample Morniga M agglutinin and/
Or hairy vetch agglutinin is formed by horizontal detectably improve of compound in conjunction with IgG4 can reflect that IgG4 is related
The Disease Activity of property disease.
4.Morniga M agglutinin and/or hairy vetch agglutinin are being prepared for reflecting the diseases related disease of IgG4
Purposes in the reagent of sick activity.
5. purposes as claimed in claim 4, which is characterized in that the reflection IgG4 diseases related Disease Activity packet
It includes: Morniga M agglutinin and/or hairy vetch agglutinin and measurement being obtained from, the diseases related patient of IgG4 is presented
Biological sample contacted, measure Morniga M agglutinin and/or hairy vetch agglutinin and formed in conjunction with IgG4
Compound level；Optionally,
Morniga M agglutinin and/or hairy vetch agglutinin and IgG4 in the biological sample compared with contrasting data
In conjunction with the level for being formed by compound, wherein relative to the contrasting data, in the sample Morniga M agglutinin and/
Or hairy vetch agglutinin is formed by horizontal detectably improve of compound in conjunction with IgG4 can reflect that IgG4 is related
The Disease Activity of property disease.
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 Morniga M agglutinin and/or hairy vetch agglutinin with
IgG4 is measured in conjunction with the level for being formed by compound by following steps, comprising:
Contact the biological sample from patient with Morniga M agglutinin and/or hairy vetch agglutinin；
B. it is formed between existing IgG4 and Morniga M agglutinin and/or hairy vetch agglutinin in the biological sample
Agglutinin-glycan compound；
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 Morniga M agglutinin and/or hairy vetch agglutinin
It deposits or is fixed on solid phase surface carrier, it is preferable that the topical carrier is latex pearl, porous flat plate or film item, receives
The form of mitron road, thin slice with two dimensional code etc..
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. one kind is for detecting and/or can quantitatively coagulate with Morniga M agglutinin and/or hairy vetch in biological sample
A kind of kit for the IgG4 that collection element combines, comprising: solid phase surface carrier, wherein the Morniga M agglutinin and/or
Hairy vetch agglutinin is deposited or is fixed on solid phase surface carrier, wherein Morniga M agglutinin and/or long pubescence
Vetch agglutinin is formed by the biology of the compound Disease Activity diseases related as reflection IgG4 in conjunction with IgG4
Marker, it is preferable that the kit further includes labeled and is reactive detection to the antibody for carrying out biological sample
Antibody, it is preferred that the topical carrier is latex pearl, porous flat plate or film item, nanotubes, the thin slice with two dimensional code etc.
Form.
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| CN113311169A (en) * | 2021-05-26 | 2021-08-27 | 重庆中元汇吉生物技术有限公司 | Kit for determining immunoglobulin G4 and preparation method thereof |
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