CN110456084A - ROBO3 autoantibody detection reagent is preparing the purposes in screening lung cancer kit - Google Patents
ROBO3 autoantibody detection reagent is preparing the purposes in screening lung cancer kit Download PDFInfo
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- CN110456084A CN110456084A CN201910895229.9A CN201910895229A CN110456084A CN 110456084 A CN110456084 A CN 110456084A CN 201910895229 A CN201910895229 A CN 201910895229A CN 110456084 A CN110456084 A CN 110456084A
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- China
- Prior art keywords
- robo3
- reagent
- lung cancer
- autoantibody
- detection
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57423—Specifically defined cancers of lung
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- 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
- G01N33/6884—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from lung
Abstract
The present invention relates to external diagnosis reagent fields, and in particular to ROBO3 autoantibody detection reagent is preparing the purposes in screening lung cancer kit.Present invention firstly discovers that the autoantibody of ROBO3 albumen is significantly higher than healthy patients in Serum of Patients with Lung Cancer.The reagent that the present invention will test ROBO3 albumen autoantibody is used to prepare screening lung cancer kit, can be realized effective screening of lung cancer.
Description
Technical field
The present invention relates to external diagnosis reagent fields, and in particular to ROBO3 autoantibody detection reagent is in preparation lung cancer sieve
Look into the purposes in kit.
Background technique
Lung cancer is that one of most common malignant tumour, morbidity and mortality are in rise year by year trend in the world, at present
Disease incidence occupies first place in the world, and seriously threatens human health and life.
Lung cancer is a kind of disease for being good at concealment, often develops to advanced stage in disease and just shows clinical symptoms, 70~
80% patients with lung cancer has been middle and advanced stage when being diagnosed to be with Lung Cancer Symptoms, and cancer cell has been spread, and misses best healing
Opportunity, five year survival rate are low.For the patients with lung cancer of early stage, giving birth to for 5 years or more for patient is greatly improved by treating in time
Deposit rate and life quality.Therefore the early diagnosis and the effective screening of progress of lung cancer are most important.
The screening of lung cancer, referring to does not have the related indication crowd of lung cancer to carry out routine physical examination those, before there is symptom
Discovery lung cancer in time.If the lung cancer molecular marker inside blood plasma can be found, for prompting clinician's early stage to patient
Relevant remedy measures or decision are taken to have great importance.
Autoantibody refers to the antibody that body generates itself organ, cell or cell component.Currently, certain albumen from
Body antibody has become the marker of lung cancer, such as: p53, NY-ESO-1, CYFRA (Tang Z-M, Ling Z-G, Wang
C-M,Wu Y-B,Kong J-L(2017)Serum tumor-associated autoantibodies as diagnostic
biomarkers for lung cancer:A systematic review and meta-analysis.PLoS ONE 12
(7):e0182117)。
ROBO3 (Ensembl:ENSG00000154134) is the member of Roundabout (ROBO) gene family, the family
Neural process is controlled to outgrowth, growth cone guidance and aixs cylinder constraint.ROBO albumen is immunoglobulin transmembrane receptor superfamily
Subfamily.SLIT albumen 1-3 is the chemocepter of a kind of secretion, is the ligand of ROBO albumen, and SLIT/ROBO interaction removes
Adjuster generation, leucocyte migration are gone back outside nerve to occur, kidney form occurs, angiogenesis and angiogenesis.Gene ROBO3 tool
There is the extracellular domain of presumption, there is 5 immunoglobulin (Ig) sample rings and 3 fibronectin (Fn) type III motifs, cross-film
Section and cytoplasmic tail with 3 conserved signal motifs: CC0, CC2 and CC3 (CC is conservative cytoplasm).With other ROBO
Family member is different, and ROBO3 lacks motif CC1.The aixs cylinder of ROBO3 Gene regulation ventral neural tube middle line is navigated.At present still
It has no the relevant report of ROBO3 albumen autoantibody, also has no its prior art relevant to lung cancer.
Summary of the invention
The purpose of the present invention is to provide the detections of the lung cancer marker and the marker of a kind of new autoantibody class
Reagent is preparing the purposes in screening lung cancer kit.
Technical solution of the present invention includes:
The reagent of detection ROBO3 albumen autoantibody is preparing the purposes in screening lung cancer kit.
The reagent of purposes as the aforementioned, the detection ROBO3 albumen autoantibody is enzyme-linked immunosorbent assay reagent
Or linked immune analysis reagent.
The reagent of purposes as the aforementioned, the detection ROBO3 albumen autoantibody is western blot reagent.
The reagent of purposes as the aforementioned, the detection ROBO3 albumen autoantibody is protein chip detection method reagent.
Purposes as the aforementioned, it is described detection ROBO3 albumen autoantibody reagent be detection human serum in ROBO3 albumen from
The reagent of body antibody.
A kind of screening lung cancer kit, it includes the reagent for detecting ROBO3 albumen autoantibody.
Kit as the aforementioned, the reagent of the detection ROBO3 albumen autoantibody are enzyme-linked immunosorbent assay examination
Agent or ELISA reagent.
The reagent of kit as the aforementioned, the detection ROBO3 albumen autoantibody is western blot reagent.
Kit as the aforementioned, the reagent of the detection ROBO3 albumen autoantibody are the examination of protein chip detection method
Agent.
Kit as the aforementioned, the reagent of the detection ROBO3 albumen autoantibody are ROBO3 albumen in detection human serum
The reagent of autoantibody.
It is of the invention it is critical that being determined that the content of ROBO3 autoantibody and the risk for suffering from lung cancer are aobvious in blood of human body
It is related, therefore the risk for suffering from lung cancer can be judged by the content of ROBO3 autoantibody in detection blood of human body, as tool
ROBO3 autoantibody means in blood of human body are surveyed in physical examination, can be using various means disclosed in the prior art, the embodiment of the present invention
It is specifically detected using enzyme-linked immune analytic method (protein chip), but is not limited only to the means, it is any to be able to detect
The method of ROBO3 autoantibody content is used equally for screening lung cancer.
The present invention provides a kind of new screening lung cancer markers and a kind of new screening lung cancer kit, can be realized lung
Effective screening of cancer；And patient can be injured very low using serum as test sample.The present invention has good application prospect.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Hereinafter " ROBO3 autoantibody " refers to " ROBO3 albumen autoantibody ".
Detailed description of the invention
Fig. 1: patients with lung cancer (LC), lung benign disease patient (DC), ROBO3 itself is anti-in normal healthy controls (NC) serum slurry
The comparison of body level.
Fig. 2: patients with lung cancer (LC) and lung's benign disease patient (DC) ROC are analyzed
Fig. 3: patients with lung cancer (LC) and normal healthy controls (NC) ROC are analyzed
Specific embodiment
The relationship of ROBO3 autoantibody and lung cancer in 1 blood plasma of embodiment
One, clinical data
Selection patients with lung cancer 30, lung's benign disease (non-malignant tumors such as tuberculosis, hamartoma) 29, normal healthy controls 29
Example, essential information are as follows:
| Essential information | Patients with lung cancer | Lung's benign disease | Normal healthy controls |
| Number | 30 | 29 | 29 |
| Age | 49.5±5.7 | 46.5±10 | 42.0±8.9 |
| Masculinity proportion | 20 (66.7%) | 12 (41.4%) | 13 (46.7%) |
Two, testing principle
HuProtTMROBO3 albumen is fixed on human protein's custom chip, and (used ROBO3 albumen is overall length egg
White, protein sequence number is ENSP00000438799), after increase serum is incubated for, ROBO3 autoantibody in serum (mainly include IgG,
IgM type antibody, there are also other types of antibody) can be in conjunction with up, cleaning removes unbonded antibody and other oroteins,
Again with anti-human IgM fluorescent marker secondary antibody (cy5 is marked, and is presented red) and anti-human igg fluorescence secondary antibody (cy3 label, green is presented)
Detection reads signal by Fluorescence Scanner, and the power of signal and the affinity and quantity of antibody are positively correlated.
Three, method
Reagent used in this part is as follows:
Specific step is as follows:
1) rewarming: chip is taken out from -80 DEG C of refrigerators, 4 DEG C of refrigerator rewarming half an hour is placed in, is subsequently placed in room temperature rewarming
15min；
2) close: the chip after rewarming fixes 14 blocks fences, and after fixing, envelope is added into each block
Liquid is closed, and is placed on side-sway shaking table, room temperature closes 3hr；
3) serum sample is incubated for: after the completion of closing, then confining liquid to the greatest extent is rapidly added preprepared serum and is incubated for
Liquid, every chip can be incubated for 14 serum samples, and the loading volume of each serum sample is 200 μ L, side-sway shaking table 20rpm, and 4 DEG C
It is incubated overnight (serum sample is firstly placed on 4 DEG C of chromatography cabinet freeze thawing, adds Incubating Solution with 1:50 dilution proportion, obtains serum Incubating Solution)；
4) it cleans: chip and chip fence being taken out together, suck sample, is then rapidly added isometric PBST, such as
This circulation for several times, guarantees when removing chip fence no cross contamination between serum sample.After removing chip fence, chip is placed in
Added with the chip cleaning box of cleaning solution, horizontal shaker, room temperature 80rpm is cleaned 3 times, each 10min；
5) secondary antibody is incubated for: chip being transferred in the incubation box that joined 3mL secondary antibody Incubating Solution, side-sway shaking table 40rpm is kept away
Light, room temperature 1hr；
6) it cleans: chip being taken out into (upper surface for paying attention to touch or scratching chip), is placed in the core added with cleaning solution
Piece cleaning box, horizontal shaker, room temperature 80rpm are cleaned 3 times, each 10min.It is cleaned 2 times with ddH2O after the completion, each 10min；
7) dry；
8) it scans: being scanned using brilliant core LuxScan 10K micro-array chip scanner；
9) data are extracted: corresponding GAL file (having recorded albumen position in chip) is opened, by chip image and
The each array of GAL file is integrally aligned, and presses automatic aligning button, is extracted data and is saved.
Four, result
The Average expression level of ROBO3 autoantibody in Plasma of The Patients With Lung Cancer is 40.0SNR (fluorescence signal relative quantification
Ratio), the Average expression level of ROBO3 autoantibody is 16.7SNR in lung's benign disease blood plasma, in normal healthy controls blood plasma
The Average expression level of ROBO3 autoantibody is 24.0SNR.Lung cancer group and lung's benign disease group (p < 0.001) and health are right
Statistically significant (Fig. 1) is compared according to group (p < 0.01).The ROC of lung cancer group and benign disease analyzes result specificity
96.6%, sensibility is 55.2% (Fig. 2)；It is 96.4% that the ROC of lung cancer group and normal healthy controls, which analyzes result specificity, sensibility
For 34.5% (Fig. 3), show that ROBO3 autoantibody can specifically distinguish lung cancer and lung's benign disease and normal healthy controls.
It can be seen from the above result that the level difference of ROBO3 autoantibody is aobvious in patients with lung cancer and the serum of non-lung cancer patient
It writes, by the level of ROBO3 autoantibody in detection serum, can achieve the purpose that screening lung cancer.
The composition and its application method one of the detection kit of the invention of embodiment 2, kit forms detection kit (14
Person-portion):
Two, kit application method
With 1 Part III of embodiment --- " detection of ROBO3 autoantibody in serum ".
Kit of the invention can be by the level of ROBO3 autoantibody in detection serum, can be with screening crowd to be checked
Suffer from the risk of lung cancer: if ROBO3 autoantibody is high (for Healthy People), the risk for suffering from lung cancer is high, if ROBO3
Autoantibody is low, then the risk for suffering from lung cancer is low.It can be used for the auxiliary diagnosis of clinical lung cancer, take relevant treatment for patient
Measure or decision provide effective foundation, and potential applicability in clinical practice is good.
Claims (10)
1. the reagent of detection ROBO3 albumen autoantibody is preparing the purposes in screening lung cancer kit.
2. purposes as described in claim 1, which is characterized in that the reagent of the detection ROBO3 albumen autoantibody is enzyme-linked
Immunosorbent adsorption test reagent or linked immune analysis reagent.
3. purposes as described in claim 1, which is characterized in that it is described detection ROBO3 albumen autoantibody reagent be
Western blot reagent.
4. purposes as described in claim 1, which is characterized in that the reagent of the detection ROBO3 albumen autoantibody is albumen
Chip detecting method reagent.
5. the purposes as described in Claims 1 to 4 is any, which is characterized in that the reagent of the detection ROBO3 albumen autoantibody
It is the reagent for detecting ROBO3 albumen autoantibody in human serum.
6. a kind of screening lung cancer kit, which is characterized in that it includes the reagent for detecting ROBO3 albumen autoantibody.
7. kit as claimed in claim 6, which is characterized in that the reagent of the detection ROBO3 albumen autoantibody is enzyme
Linked immunosorbent adsorption test reagent or ELISA reagent.
8. kit as claimed in claim 6, which is characterized in that it is described detection ROBO3 albumen autoantibody reagent be
Western blot reagent.
9. kit as claimed in claim 6, which is characterized in that the reagent of the detection ROBO3 albumen autoantibody is egg
White chip detecting method reagent.
10. the kit as described in claim 5~9 is any, which is characterized in that the detection ROBO3 albumen autoantibody
Reagent is the reagent for detecting ROBO3 albumen autoantibody in human serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910895229.9A CN110456084A (en) | 2019-09-20 | 2019-09-20 | ROBO3 autoantibody detection reagent is preparing the purposes in screening lung cancer kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910895229.9A CN110456084A (en) | 2019-09-20 | 2019-09-20 | ROBO3 autoantibody detection reagent is preparing the purposes in screening lung cancer kit |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110201517A1 (en) * | 2007-06-20 | 2011-08-18 | John Kolman | Autoantigen biomarkers for early diagnosis of lung adenocarcinoma |
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2019
- 2019-09-20 CN CN201910895229.9A patent/CN110456084A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110201517A1 (en) * | 2007-06-20 | 2011-08-18 | John Kolman | Autoantigen biomarkers for early diagnosis of lung adenocarcinoma |
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Application publication date: 20191115 |
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