CN103713125A - Kit for detecting CD137L mutation of malignant blood disease, and application of kit - Google Patents

Kit for detecting CD137L mutation of malignant blood disease, and application of kit Download PDF

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CN103713125A
CN103713125A CN201410007579.4A CN201410007579A CN103713125A CN 103713125 A CN103713125 A CN 103713125A CN 201410007579 A CN201410007579 A CN 201410007579A CN 103713125 A CN103713125 A CN 103713125A
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cd137l
kit
antibody
detecting
mutation
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CN103713125B (en
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姜文国
张树平
董秀红
许勇
杨茗
栾海云
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Binzhou Medical College
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology

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Abstract

The invention relates to compositions of a kit for detecting the CD137L mutation of a malignant blood disease, and application of the kit, and belongs to the technical field of biological medicine. The kit for detecting the CD137L mutation of the malignant blood disease is characterized by comprising a pair of antibody compositions for detecting a CD137L protein expression level. The kit can be used for detecting a tumor tissue sample of a tumor patient, then by the contrast between the tumor tissue sample and a standard graph, the type of the CD137L mutation and a mutation mechanism are determined, and the result can be used for guiding the treatment, prognosis and progression monitoring of related diseases.

Description

A kind of kit and application thereof for detection of malignant hematologic disease CD137L sudden change
Technical field
The present invention relates to a kind of composition and application thereof of the kit for detection of malignant hematologic disease CD137L sudden change, belong to biological medicine technology field.
Background technology
Tumor markers is in clinical widespread use.Its meaning mainly comprises examination health or people at highest risk, pathological grading diagnosis, prognosis judgement, therapeutic response and monitoring, detects tumor recurrence transfer.At present be mainly more extensive for the tumor markers application of digestive system tumor and genital system, as AFP, CEA, CA125 etc.
The diagnosis and treatment of neoplastic hematologic disorder also lack specific mark, by the research of the expression-form of costimulatory molecules and level introducing tumor markers, have theory innovation feature and larger realistic meaning, this research provides theoretical foundation by the early stage diagnosis and treatment for neoplastic hematologic disorder.Simultaneously also for the biomarker of target therapeutic agent is studied and screening provides important references.
Tumour be can escape immunosurveillance because of it.Tumour cell can be escaped immunity in several ways, comprising the expression that lacks costimulatory molecules.People CD137L(claims again CD137L) be to bring into play collaborative spread effect by being present in the II type memebrane protein conducted signal of surface of cell membrane, animal experiment demonstration has strengthened the cell-mediated antineoplastic immune of T.SCD137L (soluble CD137ligand) is the soluble form of CD137L, SaLih etc. extract blood serum sample healthy volunteer and the patient who suffers from malignant hematologic disease and show: acute myelocytic leukemia (acute myelocytic leukemia, AML), myelodysplastic syndrome (myelodysplastic syndrome, MDS) and non-Hodgkin lymphoma (Non-Hodgkin ' slymphoma, NHL) patients serum sCD137L level obviously improve.Preliminary proof, at AML and MDS, the patient that serum sCD137L level is high, prognosis is poor.There are 2 kinds of new montage modes in CD137L, this has produced new sCD137L.These 2 kinds of sudden changes distribute significantly different in different AML patient's hypotypes, especially lack 129 coding mutation types and progression of disease closely related.
Although found a lot of relations about CD137L expression and relevant disease progress in experiment, and the expression of intervening CD137L also has important clinical meaning to the treatment of relevant disease, but also there is no the report of the kit of special detection CD137L mutant protein expression.
Summary of the invention
The present invention aims to provide a kind of kit for detection of malignant hematologic disease CD137L sudden change, this kit can detect the neoplasmic tissue sample of tumor patient, by contrasting with standard diagram, thereby define the type of CD137L sudden change and the mechanism of sudden change, can be used in treatment and prognosis judgement and the progression of disease monitoring of instructing relevant disease.
The present invention is achieved by the following technical solutions:
For detection of a kit for malignant hematologic disease CD137L sudden change, special character is that it mainly comprises that an antagonist, SABC detect reagent and standard control collection of illustrative plates; A wherein said antagonist comprises for detection of 50-80 amino acid whose antibody of CD137L N end with for detection of 30-50 amino acid whose antibody of CD137L C end.
The above-mentioned kit for detection of malignant hematologic disease CD137L sudden change, is specifically comprised of following reagent, originates as follows, and-20 ℃ of preservations:
Component Source
The antibody (sc-11817) of CD137L C end SANTA?CRUZ
The antibody (2305-1) of CD137L N end Abcam
The goat anti-rabbit antibody of HRP mark Zhong Shan Golden Bridge
DAB chromogenic reagent Zhong Shan Golden Bridge
Confining liquid Self-control
Chromogenic reaction stops reagent Self-control
PBST eluent (10 *) Self-control
Antibody diluent Self-control
Standard immunoassay group collection of illustrative plates According to Hercep Test TM
During above-mentioned reagent forms, a described antagonist is purchased respectively from SANTA CRUZ company and Abcam company, the goat anti-rabbit antibody of described HRP mark and company of DAB chromogenic reagent buying Zi Zhongshan Golden Bridge, reagent conventional general in industry can be made or adopt to described confining liquid, chromogenic reaction termination reagent, PBST eluent (10 *), antibody diluent by oneself, and described standard immunoassay group collection of illustrative plates is according to Hercep Test tMand obtain.(note: mentioned reagent source is not limited to these companies)
A kind of kit for detection of malignant hematologic disease CD137L sudden change of the present invention, the pair of primers combination providing is for detection of CD137L protein expression level, by secondary computer photo, analyze and contrast with standard diagram, determine type and mechanism that splice mutation occurs, instruct malignant hematologic disease and express and change relevant clinical treatment of curing the disease to CD137L.
Accompanying drawing explanation
The impact of the Mabthera of Fig. 1 variable concentrations on the propagation of Raji, Raji/C and Raji/CD137L cell;
Fig. 2 Raji, Raji/C and the impact of Raji/CD137L cell on lymphocytic propagation;
The impact that Fig. 3 Raji, Raji/C and Raji/CD137L cell discharge lymphocyte IL-2;
Fig. 4 standard immunoassay group collection of illustrative plates.
The invention provides a kind of kit for detection of malignant hematologic disease CD137L sudden change, this kit can detect the neoplasmic tissue sample of tumor patient, by contrasting with standard diagram, thereby define the type of CD137L sudden change and the mechanism of sudden change, can be used in treatment and prognosis judgement and the progression of disease monitoring of instructing relevant disease.
Antibody combination N end antibody in the present invention is for detecting 68 amino acid whose antibody of CD137LN end; C end antibody is for detecting 42 amino acid whose antibody of CD137LC end.
Criterion and the method for protein level have been disclosed in certain embodiments, when the software for calculation parameter designing adopting in invention changes, the accumulation optical density value obtaining (integrated optical density, IOD) can change, and can have influence on the judgement of result.In testing process, to require to carry out standard color value in strict accordance with standard diagram (Fig. 4), to avoid deviation.The image processing software that can use non-kit to recommend calculates IOD value, but standard color value should be consistent.
Find in certain embodiments, transfection CD137L has synergy to the treatment of disease, illustrates that the disappearance of CD137L and the progress of disease are closely related, is the important target spot for the treatment of correlative diseases.For the further detection of generation mechanism, can instruct the enforcement of clinical chemotherapy scheme.For the sCD137L patient who directly expresses outside transporte to cells, can supplement costimulatory signal, improve result for the treatment of; For the sCD137L patient who is produced by matrix metalloproteinase (matrix metalloproteinase, MMP) cutting, take to suppress the active individualized treatment scheme of MMP.
Kit of the present invention is specifically comprised of following reagent, originates as follows, and-20 ℃ of preservations.
Component Source
The antibody (sc-11817) of CD137L C end SANTA?CRUZ
The antibody (2305-1) of CD137L N end Abcam
The goat anti-rabbit antibody of HRP mark Zhong Shan Golden Bridge
DAB chromogenic reagent Zhong Shan Golden Bridge
Confining liquid Self-control
Chromogenic reaction stops reagent Self-control
PBST eluent (10 *) Self-control
Antibody diluent Self-control
Standard immunoassay group collection of illustrative plates According to Hercep Test TM
In a further embodiment, find, in other non-diseases in the blood system, also to find that similar gene mutation occurs CD137L, as the tumour of bottleneck throat.Still there is in actual applications important clinical meaning.
Embodiment
Mode by embodiment is further illustrated to the present invention below, but therefore do not limit the present invention in described scope of embodiments.
Embodiment 1, the variation of transfectional cell to Mabthera susceptibility
Mabthera is that global first is approved for the monoclonal antibody of clinical treatment non-Hodgkin lymphoma (NHL), and Mabthera is inhibited to the growth of Raji cell, and has concentration dependent and time dependence.This research show Mabthera to the inhibiting effect of Raji and Raji/C without obviously difference (Fig. 1), and can produce stronger inhibiting effect for the Raji/CD137L cell of transfection CD137L, to Mabthera Increased sensitivity, at concentration > 2.5mg/mL, rate of increase rate is significantly lower than Raji and Raji/C.
The impact of embodiment 2, transfectional cell human peripheral blood lymphocyte activity
In order to detect the lymphopoietic impact of transfection CD137L cell human peripheral blood, adopt lymphocyte and Raji, Raji/C and the Raji/CD137L cell co-cultivation of different effect/target ratios, adopt CCK-8 cell proliferation detecting kit to measure and respectively organize cytoactive (Fig. 2), the Raji cell that shows transfected with human CD137L can significantly promote lymphocytic propagation, and along with the increase of effect/target ratio, promote proliferation activity stronger (P < 0.01).
The impact that embodiment 3, transfectional cell discharge the factor
IL-2 is important effector cell's factor, and it can activate other T lymphocyte and bone-marrow-derived lymphocyte, and stimulates NK cell proliferation, has antineoplastic action.This research finds that transfection Raji/CD137L can significantly promote peripheral blood lymphocyte to discharge IL-2(P < 0.01), and along with the increase of effect/target ratio, effect enhancing (Fig. 3).
Embodiment 4, the CD137L expression in lymphoma tissue
What adopt the antibody test of N end is the expression of total CD137L, and C end antibody mainly detects the expression of film mating type CD137L, therefore overall IOD value N end mensuration group is greater than C end mensuration group.Result shows, CD137L expression and age, sex and histological type etc. are without significant correlation, and just HD patient N end CD137L expression is compared with NHL patient Lve Gao (table 1).In addition, to same tissue, adopt the IOD value of the CD137L that two kinds of different antibodies measure respectively, find that both IOD values there is no significant correlation (P>0.05).This illustrates that the ratio that different patient CD137L changes sCD137L into is random.
Table 1 tissue microarray assay CD137L antigen expression in lymphoma tissue
Figure BDA0000454476020000051
Embodiment 5, CD137L are in throat cancer and the expression of the other normal structure of cancer and the relation between pathological factor
We have carried out simple statistics to positive rate.In addition, adopt Image-Pro Plus6.0 image analysis software batch program to calculate the IOD value of each interlacing point.Then IOD value is carried out to statistical analysis as measurement data.Statistics shows, its expression and age, sex and tumor location, without significant correlation, have certain correlativity with histological type and tumor grade.Meanwhile, we also compare two kinds of different antibodies and measure CD137L expressions, found that both there is no significant correlation (P>0.05).This explanation is different throat cancer patients, and the ratio that splice mutation occurs CD137L is random, this with CD137L at result of study of other diseases expression conform to (table 2).
Table 2 tissue microarray assay CD137L antigen is in the expression of throat cancer and the other normal structure of cancer
Figure BDA0000454476020000061

Claims (5)

1. for detection of a kit for malignant hematologic disease CD137L sudden change, it is characterized in that, comprise an antagonist combination that detects CD137L protein expression level for adopting.
2. a kind of kit for detection of malignant hematologic disease CD137L sudden change as claimed in claim 1, be characterised in that, described antibody combination comprises that can detect CD137L N holds 50-80 amino acid whose antibody and can detect 30-50 amino acid whose antibody of CD137L C end.
3. a kind of kit for detection of malignant hematologic disease CD137L sudden change as claimed in claim 1, is characterised in that specifically and is comprised of following reagent:
The antibody of CD137L C end
The antibody of CD137L N end
The goat anti-rabbit antibody of HRP mark
DAB chromogenic reagent
Confining liquid
Chromogenic reaction stops reagent
PBST eluent (10 *)
Antibody diluent
Standard immunoassay group collection of illustrative plates.
4. the application of the kit described in the arbitrary claim of claim 1-3 in detecting malignant tumour.
5. the application of the kit described in the arbitrary claim of claim 1-3 in detecting hematologic malignancies.
CN201410007579.4A 2014-01-08 2014-01-08 A kind of for detect malignant hematologic disease CD137L suddenly change kit and application Expired - Fee Related CN103713125B (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006088464A2 (en) * 2005-02-15 2006-08-24 Gtc Biotherapeutics, Inc. A method of using an anti-cd137 antibody as an agent for radioimmunotherapy or radioimmunodetection
CN1844149A (en) * 2005-04-07 2006-10-11 苏州大学 Monoclonal antibody against human 4-1BBL and its use
CN1880446A (en) * 2005-06-16 2006-12-20 VIRxSYS公司 Antibody complexes
CN102174522A (en) * 2011-02-24 2011-09-07 杭州师范大学 Preparation method of protein 4-1BBL

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006088464A2 (en) * 2005-02-15 2006-08-24 Gtc Biotherapeutics, Inc. A method of using an anti-cd137 antibody as an agent for radioimmunotherapy or radioimmunodetection
CN1844149A (en) * 2005-04-07 2006-10-11 苏州大学 Monoclonal antibody against human 4-1BBL and its use
CN1880446A (en) * 2005-06-16 2006-12-20 VIRxSYS公司 Antibody complexes
CN102174522A (en) * 2011-02-24 2011-09-07 杭州师范大学 Preparation method of protein 4-1BBL

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KATSUNORI KASHIMA, ET AL.: "Screening of BRCA1 Mutation Using Immunohistochemical Staining with C-Terminal and N-Terminal Antibodies in Familial Ovarian Cancers", 《JPN. J. CANCER RES.》, vol. 91, 30 April 2000 (2000-04-30), pages 399 - 409, XP002573682 *
宋敏: "CD137L在急性髓细胞白血病中的突变表达及临床意义研究", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑》, no. 4, 15 April 2013 (2013-04-15) *

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