CN106680513A - Application of antibody for resisting CD4 (Cluster of Differentiation 4) and antibody for resisting CXCR5 (C X Chemokine Receptor 5) in preparing reagent for assessing chronic graft versus host disease - Google Patents
Application of antibody for resisting CD4 (Cluster of Differentiation 4) and antibody for resisting CXCR5 (C X Chemokine Receptor 5) in preparing reagent for assessing chronic graft versus host disease Download PDFInfo
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- CN106680513A CN106680513A CN201710104048.0A CN201710104048A CN106680513A CN 106680513 A CN106680513 A CN 106680513A CN 201710104048 A CN201710104048 A CN 201710104048A CN 106680513 A CN106680513 A CN 106680513A
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses application of an antibody for resisting CD4 (Cluster of Differentiation 4) and an antibody for resisting CXCR5 (C X Chemokine Receptor 5) in preparing a reagent for assessing a cGVHD (Chronic Graft Versus Host Disease). The invention aims to provide the antibody for resisting CD4 and the antibody for resisting CXCR5 as measurement indexes for assessing cGVHD severity so as to guide clinical therapy. On the basis of the research, the inventor utilizes a flow cytometry method for detecting CD4+ CXCR5+circulation Tfh cells in peripheral blood of an able-bodied person and a postoperative patient with allogeneic hematopoietic stem cell transplantation, and the disease severity of a disease is determined through a simple, convenient and effective cellular immunology method so as to guide clinical therapy.
Description
Technical field
The present invention relates to the new application of the antibody of the antibody of anti-CD4 and anti-CXCR5, and in particular to the antibody of anti-CD4 and anti-
Application of the antibody of CXCR5 in the assessment CGVHD conditions of patients order of severity.
Background technology
CGVHD (cGVHD) is that one kind shows as polymorphy autoimmune disease sample syndrome, is led
Cause the multiple organs such as oral cavity, eyes, skin, lung and liver tissue chronic inflammation and fibrillatable sample pathology damage；Allogeneic is done
The incidence of disease of cell transplantation postoperative patient cGVHD is up to 30-70%, 5 years survival rate about 30-50%, is that allogeneic is dry thin
The main cause of the posttransplantation major complications of born of the same parents and non-Recurrent death.
The pathogenesis of CGVHD relates generally to chronic inflammation, cellular immunity, humoral immunity and fiber
Change, the Most patients after Allogeneic Hematopoietic Stem Cell Transplantation in 1 year have a clinical manifestation of different degrees of cGVHD, and in
Severe cGVHD has a strong impact on the quality of life of patient.Diagnosis and condition assessment at present for cGVHD mainly works according to NIH
The standard of group, is shown in Table 1：NIHcGVHD organs or position scoring and integrating system
Table 1
Note：Lung's scoring will simultaneously take into account symptom and pulmonary function test, when both are inconsistent, select the high person of scoring.Choosing
PFS is selected more preferably, but if DLCO can not be checked, it should check FEV1.The scoring of FEV1 and DLCO is as follows：＞ 80%
=1,70~79%=2,60~69%=3,50~59%=4,40~49%=5, ＜ 40%=6, PFS=
FEV1 scoring+DLCO scorings, scope is at 2~12 points.
At least one chronic GVHD diagnostic characteristic and a distinctive performance, and exclude infection and other reasonses such as exist
The relevant hypoplastic nails of onychomycosis, herpe simplex, oral candida albicans infection, drug poisoning etc. in cGVHD complication.It is right
It is subjective and complicated loaded down with trivial details although the assessment mode system is comprehensively in the assessment of the order of severity of cGVHD, to medical matters
The clinical experience of personnel requires higher.Currently cGVHD pathogenesis is still not clear, lacks the biology of medical diagnosis on disease assessment
Mark so that the diagnosis of cGVHD and condition assessment are still a great challenge.
At present the medicine for cGVHD mainly includes cortin and immunodepressant.Existing therapeutic modality with
Non-selective suppression T cell is removed based on B cell, and non-targeted treatment causes malignant disease recurrence, repeatedly severe infections, secondary
Second tumour and cGVHD protracted inflammations repeatedly, have a strong impact on the quality of life of patient.Clinically to cortin and exempt from present
The dosage adjustment of epidemic disease inhibitor lacks the direct biological indicator of curative effect evaluation mainly according to NIH standards of grading, has a strong impact on trouble
The treatment of person.
In sum, although our diagnosis to cGVHD and treatment have certain understanding, it is a lack of applying direct indicator
Disease is assessed by the change level of immunocyte, this is most important for direction of medication usage, assessment curative effect of medication, and it develops work
Make imperative.
Numerous studies confirm B cell as the antigen presenting cell in immune response chain and effector cell, sending out in cGVHD
There is important function in hair tonic exhibition.B cell is activated and function is implemented to depend on the auxiliary of T cell, and research in recent years finds, positioning
In lymph follicle, the folliculus helper T lymphocyte of continuous expression CXCR5 is the main auxiliary cell of B cell, is starting humoral immunity
Play a significant role in response.Tfh cells are most earlier than 2000 by Sehaerli and Breitfeld etc. almost simultaneously in tonsillotome
It is middle to find simultaneously to be named as tfh cells, 2008, King etc. determine it be in lymphoid tissue most important effector T cell subgroup it
One, in being distributed mainly on lymphoid tissue, cell overexpression in lymphoid tissue can then cause autoimmune disease, and with
The target organ involvement order of severity has much relations, and 2016, EdouardForcade etc. had found active stage cGVHD patient CD4+
CXCR5+ circulation Tfh cell proportions are significantly higher than Normal group, and cGVHD patient's circulation Tfh cells are gone back to the nest to Secondary cases and drenched
The quantity of bar organ significantly increases.
Researcher's research discovery of the present invention, low expression CD4+CXCR5 in middle severe cGVHD patient peripheral blood lymphocyte
+ circulation Tfh cells, this group of cells by Flow cytometry out, and can have extremely strong with existing disease severity
Correlation, the immunocyte out-of-balance conditions that can be directly acquainted with patient's body, for the treatment of middle severe cGVHD has high guidance
Meaning.
The content of the invention
It is an object of the invention to provide the new application of the antibody of the antibody of anti-CD4 and anti-CXCR5.
To achieve these goals, present invention employs technical scheme below：The antibody of the antibody of anti-CD4 and anti-CXCR5
The application in reagent in assessment CGVHD is prepared.
The antibody of the anti-CD4 is the monoclonal antibody of the anti-CD4 of commercially available monoclonal antibody or in vitro culture secretion
The monoclonal antibody that hybridoma is obtained.
The antibody of the anti-CD4 is the monoclonal antibody purchased from ebiscience or Biolegend companies.
4th, application according to claim 1, it is characterised in that：The antibody of the anti-CXCR5 is commercially available monoclonal
The monoclonal antibody that the hybridoma of the monoclonal antibody of antibody or the anti-CXCR5 of in vitro culture secretion is obtained.
The antibody of the anti-CXCR5 is the monoclonal antibody purchased from ebiscience or Biolegend companies.
Researcher of the present invention has found to can detect that expression has in cGVHD patient and human peripheral lymphocytes
The circulation Tfh cells of the significant surface molecular such as CD4, CXCR5.Our research has shown that CD4+CXCR5+ circulation Tfh cells exist
Ratio is significantly reduced in the lymphocyte of middle severe cGVHD patient.If CD4+CXCR+ circulations in patient's lymphocyte after transplanting
The expression of Tfh cells reduces obvious, points out patient to be likely to occur middle severe rejection.Therefore detection CD4+CXCR5+ circulates Tfh
Cell can be used as immunocyte mark, the cGVHD of severe in correct judgement, the abnormal immunocyte state of direct reaction,
For clinical treatment has important directive significance.
It is an object of the invention to provide anti-CD 4 antibodies and anti-CXCR5 antibody refer to as the measurement for evaluating the cGVHD orders of severity
Mark, guiding clinical treatment.Study based on more than, this research inventor detects that normal person and allogene are made with flow cyctometry method
CD4+CXCR5+ circulations Tfh cells in hemocytoblast post-transplantation peripheral blood in patients, by simple and effective cellular immunology side
Method determines the coincident with severity degree of condition of disease, guiding clinical treatment.
Description of the drawings
Fig. 1 a~Fig. 1 c are that streaming result shows normal person and Allogeneic Hematopoietic Stem Cell Transplantation postoperative patient periphery blood strangury
In bar cell CD4+CXCR5+ circulation Tfh cells ratio schematic diagram wherein, Fig. 1 a：HC healthy control groups；Fig. 1 b：mild
CGVHD is slight cGVHD；Fig. 1 c：Severe cGVHD in moderate＆severve GVHD.
Fig. 2 is CD4+CXCR5+ circulation Tfh cell proportions slight cGVHD patient and middle heavy after normal healthy controls person, transplanting
Ratio schematic diagram in degree cGVHD peripheral blood in patients.(P is equal<0.05).
Specific embodiment
1st, all cGVHD patients of following examples disease layering all in accordance with NIH cGVHD organs or position scoring and
Integrating system (sees above table 1).
NIH whole world cGVHD integrating systems are number and each organ/portion of the NIH working groups according to afflicted organ or position
The global cGVHD integrating systems that the position involvement order of severity is worked out, refer to table 2 below：
Table 2
| Organ/number of sites | Slight cGVHD | Moderate cGVHD | Severe cGVHD |
| 1 | Integration 1 | Integration 2 | Integration 3 |
| 2 | Integration 1 | Integration 2 | Integration 3 |
| 3 | Integration 1 | Integration 3 | |
| Lung is involved | Integration 1 | Integration 2 |
Note：0=is asymptomatic for integration, integrates 1=light symptoms, integrates 2=moderate symptoms, integrates 3=serious symptoms.Slightly
CGVHD involves 1 or 2 organs (except lung), and the maximum integration of involved is 1 in each organ, and moderate cGVHD involves 1 or 2
Individual organ, or 3 and more maximum organs integrated as 1, or lung is 1 by iterated integral.Severe cGVHD involves at least one
The order of severity is 3 organ, or lung is 2 by iterated integral.
2nd, CD4+CXCR5+ circulates the concrete detection method of Tfh cells：
Using Flow Cytometry detection CD4+CXCR5+ circulation Tfh cells, its operating process is as follows：
Peripheral blood 100ul is taken, lymphocyte is about 1~3 × 105It is individual, mark fluorescent antibody CD4-FITC
(ebioscience) 5ul/test, CXCR5-APC-cy7 (biolegend) 5ul/test, is vortexed and mixes, the incubation of room temperature lucifuge
25 minutes, 1 × erythrocyte cracked liquid 3mL is added, slightly mixed, room temperature avoid light place 10 minutes, 300g centrifugation 5min abandon supernatant
Liquid, adds 1ml PBS, 300g centrifugation 5min, abandons supernatant, adds machine testing on 400ul PBS.Using BD CantoII streamings
The cell expression ratio of CD4+CXCR5+ in cell instrument detection lymphocyte.
The study show that：CD4+CXCR5+ circulates Tfh cells low expression in middle severe cGVHD peripheral blood in patients, hence it is evident that
Less than Normal group and slight cGVHD patient, the determination of centering severe cGVHD patient has great importance.
This research circulates the expression rate of Tfh cells by Flow cytometry each group CD4+CXCR5+, and each group detection is shown
Intention is shown in Fig. 1 a~Fig. 1 c (enclose cell colony in three groups of figures in square frame and be CD4+CXCR5+ circulation Tfh cells).
This research detects altogether 29 normal persons, severe cGVHD patient peripheral's blood strangury in 35 slight cGVHD patients and 36
The ratio of Tfh cells is circulated in bar cell.Positive boundary's point is defined as with the mean-SD less than normal person.
This research FACS methods have detected CD4+CXCR5+ and circulate Tfh cells in each group patient and the positive of normal person
Rate, is shown in Table 3：CD4+CXCR5+ circulates the comparison of Tfh cells the positive expression rate in different order of severity cGVHD patients.Using
X2Each group of data is compared in inspection, as a result shows, it is notable that CD4+CXCR5+ circulates positive rates of the Tfh in middle severe cGVHD patient
Higher than normal person and slight cGVHD patient, P is equal<0.05.
Table 3
Claims (5)
1. the application in reagent of the antibody of the antibody of anti-CD4 and anti-CXCR5 in assessment CGVHD is prepared.
2. application according to claim 1, it is characterised in that：The antibody of the anti-CD4 be commercially available monoclonal antibody or
The monoclonal antibody that the hybridoma of the monoclonal antibody of the anti-CD4 of in vitro culture secretion is obtained.
3. application according to claim 2, it is characterised in that：The antibody of the anti-CD4 be purchased from ebiscience or
The monoclonal antibody of Biolegend companies.
4. application according to claim 1, it is characterised in that：The antibody of the anti-CXCR5 is commercially available monoclonal antibody
Or the monoclonal antibody that the hybridoma of the monoclonal antibody of the anti-CXCR5 of in vitro culture secretion is obtained.
5. application according to claim 4, it is characterised in that：The antibody of the anti-CXCR5 be purchased from ebiscience or
The monoclonal antibody of Biolegend companies.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710104048.0A CN106680513A (en) | 2017-02-24 | 2017-02-24 | Application of antibody for resisting CD4 (Cluster of Differentiation 4) and antibody for resisting CXCR5 (C X Chemokine Receptor 5) in preparing reagent for assessing chronic graft versus host disease |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710104048.0A CN106680513A (en) | 2017-02-24 | 2017-02-24 | Application of antibody for resisting CD4 (Cluster of Differentiation 4) and antibody for resisting CXCR5 (C X Chemokine Receptor 5) in preparing reagent for assessing chronic graft versus host disease |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406775A (en) * | 2018-10-12 | 2019-03-01 | 东莞市暨科生物科技有限公司 | Autoimmune disease patient's immune function assesses kit and appraisal procedure |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130150252A1 (en) * | 2011-12-13 | 2013-06-13 | Sequenta, Inc. | Detection and measurement of tissue-infiltrating lymphocytes |
| CN103487572A (en) * | 2013-10-08 | 2014-01-01 | 北京大学人民医院 | Application of T follicular helper lymphocyte (Tfh) in autoimmune disease early warning |
-
2017
- 2017-02-24 CN CN201710104048.0A patent/CN106680513A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130150252A1 (en) * | 2011-12-13 | 2013-06-13 | Sequenta, Inc. | Detection and measurement of tissue-infiltrating lymphocytes |
| CN103487572A (en) * | 2013-10-08 | 2014-01-01 | 北京大学人民医院 | Application of T follicular helper lymphocyte (Tfh) in autoimmune disease early warning |
Non-Patent Citations (1)
| Title |
|---|
| DAVID A. KNORR等: "Stem Cell Source Dependent Differences in Reconstituting Peripheral T Follicular Helper Cells Following Hematopoietic Cell Transplantation, Their Association with Chronic Gvhd and Role in Response to Influenza Vaccination", 《BLOOD》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109406775A (en) * | 2018-10-12 | 2019-03-01 | 东莞市暨科生物科技有限公司 | Autoimmune disease patient's immune function assesses kit and appraisal procedure |
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