CN109655624A - A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit - Google Patents
A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit Download PDFInfo
- Publication number
- CN109655624A CN109655624A CN201910109792.9A CN201910109792A CN109655624A CN 109655624 A CN109655624 A CN 109655624A CN 201910109792 A CN201910109792 A CN 201910109792A CN 109655624 A CN109655624 A CN 109655624A
- Authority
- CN
- China
- Prior art keywords
- cancer
- kit
- antibody
- predicting
- microballoon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- G01N33/6893—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
- 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
- G01N33/57488—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 involving compounds identifable in body fluids
-
- 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
- G01N33/6854—Immunoglobulins
Abstract
The present invention provides a kind of for predicting the Serology biological marker of cancer immunotherapy effect, belongs to biomedicine technical field.Biomarker of the present invention is IL-1RA.Experiment shows that the effective patient of immunologic test point blocking treatment, IL-1RA content exists and significantly reduces in pretherapy and post-treatment serum.The present invention also provides the application prepared using above-mentioned biomarker for predicting the kit of cancer immunotherapy effect, the kit includes the phycoerythrin of the coding microball for being coated with IL-1RA capture antibody, IL-1RA the detection antibody and marked by streptavidin of biotin labeling.The prediction that cancer immunity checkpoint blocking treatment effect is carried out using IL-1RA biomarker provided by the invention or its kit being prepared, is had the advantages that detection is quick, accurate, expense is low etc., had a extensive future.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of for predicting the mark of cancer immunotherapy effect
Object and its preparation method of application, kit and kit.
Background technique
Rapid development and Cross slot interference with related disciplines such as oncology, immunology and molecular biology, tumour are exempted from
The research of epidemic disease treatment achieves breakthrough, gradually becomes most promising one of the research direction of therapeutic field of tumor.
Immunization therapy becomes another important antineoplaston means after operation, radiotherapy and chemotherapy.Tumour at present
Immunization therapy mainly includes vaccine, immunologic test point inhibitor for treating, adoptive immunity cell therapy, cytokine therapy etc.,
Wherein immunologic test point Blocking therapy is attracted attention with its significant clinical efficacy.Immunologic test point is in human immune system
The molecule for playing " brake " and protective effect, the purpose is to prevent T cell excessive activation and lead to inflammation damnification etc..Tumour cell is then
Inhibit Human immune responses by raising the expression of immunologic test point molecule using this characteristic, escape immunosurveillance and kill
Wound.Study and apply at present widest immunologic test point molecule includes CTLA4 and PD-1/PD-L1.Immunologic test point inhibits
Agent therapy uses the specific antibody of CTLA4 or PD-1 or PD-L1 to be injected into tumor patient body, so that tumour no longer has
The ability for escaping immune system attack, to reactivate T cell to the immune response effect of tumour cell.Currently, the U.S. is eaten
Product and Drug Administration (FDA) have approved panimmunity checkpoint inhibitor for variety classes solid tumor (melanoma, lung
Cancer, gastric cancer, colorectal cancer etc.) and hematologic malignancies treatment.
Immunologic test point inhibitor for treating has had more and more patients by nearly research in 30 years and clinical application
Therefrom benefit, however since only some patient is more sensitive to immunologic test point Blocking therapy, filter out predictable treatment
The biomarker of effect and adverse events is a vital challenge.The biology mark of outcome prediction more commonly used at present
Will object includes the expression quantity of PD-1/PD-L1, microsatellite instability (MSI), mispairing gene repair lacks (MMR), Tumor mutations are born
Lotus (TMB), tumor infiltrating lymphocyte (TILs) etc..For example, multinomial clinical research shows that the raised tumour of PD-L1 expression quantity is suffered from
Curative effect is better using PD-L1 Antybody therapy by person.In addition, if occurring mispairing gene repair missing in the carcinoma of the rectum and micro- defending
The unstable patient of star, it is more using benefiting after PD-1/L1 inhibitor.Tumor mutations load is also the relevant factor of curative effect, is faced
Bed is research shows that high expression of the Tumor mutations load in malignant mela noma, lung cancer and colon cancer is treated with the clinical of immunization therapy
Effect is positively correlated.However, current various biomarkers have its limitation, facing for patient can not be predicted to entirely accurate
Bed curative effect and benefit situation.Therefore, the biomarker for exploring and developing prediction cancer immunotherapy effect has important face
Bed meaning.
Tumour related inflammation is the research hotspot in tumour immunity field in recent years.Inflammation is referred to as the eighth-largest of malignant tumour
Biological property has played important function in each stage of tumour.In recent years, using interleukin-1 (IL-1) family as representative
Effect of inflammatory factor during tumor development have become the hot spot for research.Wherein IL-1 α in IL-1 subtribe,
The researchs such as IL-1 β and IL-1RA (also known as interleukin-1 receptor antagonist) are more deep.IL-1 family member be congenital immunity and
The central mediators of inflammation are a kind of pleiotrophic cytokines with a variety of locally and systemically effects.Most of IL-1 families cell
The factor has proinflammatory activity (such as IL-1 α, IL-1 β), and few members (such as IL-1RA) have anti-inflammatory effect.It is sent out in tumour
They show different biological functions during exhibition.IL-1 α from necrotic tumor cells may promote carcinous inflammation
Reaction；IL-1 β may play a role in advanced tumor, for example, tumor microenvironment generate IL-1 β can by with angiogenesis
The factor (VEGF) interaction driving angiogenesis；IL-1 β may also participate in the differentiation of Th17 and the generation of relevant cell factor.
IL-1RA can combine closely with interleukin 1 receptor I type (IL-1R1), to block IL-1 α and IL-1 β and its receptor knot
It closes, so that IL-1 be blocked to play a role.Research there is no to prompt at present, IL-1RA can be used as immunotherapy of tumors outcome prediction
Serology biological marker.
Liquid-phase chip, also referred to as microsphere suspending chip (suspension array, liquid chip), are to be based on
The Novel biological chip technology platform of xMAP (flexible Multi Analyte Profiling) technology, it is different glimmering
On the microballoon of pumped FIR laser carry out Ag-Ab, enzyme-substrate, ligand-receptor association reaction and nucleic acid hybridization reaction, by it is red,
Green two beams laser detects microballoon coding respectively and reporter fluorescence is qualitative and quantitative to achieve the purpose that, can be complete in a reacting hole
It is the high-throughput Molecular Detection skill of a new generation after genetic chip, protein chip at up to 100 kinds of different biologicallies
Art platform.Ten years so far, the whole world have hundreds of sets based on the detection platform of xMAP technology for immunology, albumen
The fields such as matter, detection of nucleic acids, gene studies, the technology have become a kind of new proteomics and genomics research tool,
It is also earliest by the biochip technology that can be used for clinical diagnosis of U.S. Food and Drug Administration (FDA) certification.
Summary of the invention
The Serology biological marker that the purpose of the present invention is to provide a kind of for predicting cancer immunotherapy effect and
It is preparing the application in the kit for predicting cancer immunotherapy effect.
The present invention provides a kind of for predicting the Serology biological marker of cancer immunotherapy effect, the serology
Biomarker is IL-1RA.
Preferably, the cancer includes gastroenteric tumor.
Preferably, the gastroenteric tumor includes in the cancer of the esophagus, gastric cancer, colorectal cancer, liver cancer, cancer of pancreas and cholangiocarcinoma
It is one or more kinds of.
The present invention provides above-mentioned Serology biological markers to prepare the reagent for predicting cancer immunotherapy effect
Application in box.
The present invention provides a kind of for predicting the liquid phase chip reagent box of cancer immunotherapy effect, in the kit
Including being coated with the coding microball of IL-1RA capture antibody, the IL-1RA detection antibody and marked by streptavidin of biotin labeling
Phycoerythrin.
Preferably, the clone number of the IL-1RA capture antibody is 998A2A2.
Preferably, the clone number of the IL-1RA detection antibody is A71B6D11.
Preferably, the coding microball includes carboxyl microballoon.
Preferably, the biotin includes N- carboxylic succinimide activated biotin.
The present invention also provides the preparation methods of above-mentioned liquid phase chip reagent box, include the following steps:
(1) IL-1RA capture antibody and coding microball are coupled, obtain the coding for being coated with IL-1RA capture antibody
Microballoon；
(2) biotin is connected on IL-1RA detection antibody, obtains the IL-1RA detection antibody of biotin labeling；
The step (1) and (2) are limited without time sequencing.
The utility model has the advantages that the present invention provides a kind of for predicting the Serology biological marker of cancer immunotherapy effect,
The biomarker is IL-1RA.Experiment shows to the effective patient of immunologic test point blocking treatment, pretherapy and post-treatment blood
IL-1RA content exists and significantly reduces in clear.The present invention also provides prepared using above-mentioned biomarker for predicting that cancer is exempted from
The application of the kit of epidemic disease therapeutic effect, the kit include coding microball, the biotin for being coated with IL-1RA capture antibody
The phycoerythrin of IL-1RA the detection antibody and marked by streptavidin of label.Utilize IL-1RA biological marker provided by the invention
Object or its kit being prepared carry out the prediction of cancer immunity checkpoint blocking treatment effect, have detection quick, quasi-
Really, the advantages such as expense is low, have a extensive future.
Detailed description of the invention
Fig. 1 is the standard curve schematic diagram for detecting biomarker IL-1RA in serum；
Fig. 2 is that liquid phase chip reagent box detects IL-1RA changes of contents difference before and after patients with gastrointestinal carcinoma immunization therapy
Figure；
Fig. 3 is to be controlled using the content difference of IL-1RA biomarker before and after immunotherapy of patients patients with gastrointestinal carcinoma
Receiver operating curve's (ROC curve) of the validity prediction for the treatment of；
Fig. 4 be Tumor Patient Before and After Treatment serum in IL-1RA biomarker variation [changing value definition: (after treatment-
Before treatment) before/treatment] to the recurrence-free survival rate effect being classified and predicted after the cancer of the esophagus and colorectal cancer patients treatment
Fruit.
Specific embodiment
The present invention provides a kind of for predicting the Serology biological marker of cancer immunotherapy effect, the serology
Biomarker is IL-1RA.In the present invention, the cancer preferably includes gastroenteric tumor.The gastroenteric tumor preferably wraps
Include one or more of the cancer of the esophagus, gastric cancer, colorectal cancer, liver cancer, cancer of pancreas and cholangiocarcinoma.The present invention through experiments, it was found that,
The content of IL-1RA is related with immunologic test point blocking treatment effect in cancer patient's serum sample sheet, and cancer patient is used alone and controls
The changes of contents for treating IL-1RA in the serum sample of front and back can predict the effect of cancer immunity checkpoint blocking treatment.Based on
IL-1RA Serology biological marker predicts that the had accuracy rate of the effect of immunologic test point blocking treatment is high, it is more square to implement
Just, the advantages of cost is relatively low has broad application prospects.
The present invention provides above-mentioned Serology biological markers to prepare the reagent for predicting cancer immunotherapy effect
Application in box.In the present invention, the application includes being prepared based on above-mentioned biomarker arbitrarily with spy
The opposite sex detects the kit of above-mentioned biomarker function.
The present invention provides a kind of for predicting the kit of cancer immunotherapy effect, includes coating in the kit
There is the algae red egg of the coding microball of IL-1RA capture antibody, IL-1RA the detection antibody and marked by streptavidin of biotin labeling
It is white.In the present invention, the clone number of the IL-1RA capture antibody is preferably 998A2A2.Gram of the IL-1RA detection antibody
Grand number preferably A71B6D11.The coding microball is preferably carboxyl microballoon.The biotin is preferably N- carboxylic succinimide
Activated biotin.In the present invention, the kit is to be measured first with the coding microball capture for being coated with IL-1RA capture antibody
Then IL-1RA in sample utilizes the phycoerythrin pair of IL-1RA the detection antibody and marked by streptavidin of biotin labeling
Obtained IL-1RA is captured to be quantified.
The present invention is based on liquid-phase chip technology, the IL-1RA biomarker in serum can quickly be examined by developing
The liquid phase chip reagent box of survey.The kit has many advantages, such as that without side-effects, susceptibility is high, detection is quick, reproducible.The liquid
The preparation method of phase chip agent box is simple and reliable, stability is good.
The present invention also provides the preparation methods of mentioned reagent box, include the following steps:
(1) IL-1RA capture antibody and coding microball are coupled, obtain the coding microball for being coated with IL-1RA capture antibody；
(2) biotin is connected on IL-1RA detection antibody, obtains the IL-1RA detection antibody of biotin labeling；
The relationship of sequencing is not present between the step (1) and (2).
IL-1RA capture antibody and coding microball are coupled by the present invention, and the coding for obtaining being coated with IL-1RA capture antibody is micro-
Ball.In the present invention, the method for the coupling preferably includes following steps:
A. carboxyl microballoon is taken, 15~25s of microsphere suspensions is vibrated with vortex oscillator, is uniformly mixed microballoon；
B. the carboxyl microballoon 0.5 × 10 after taking concussion6~1.5 × 106It is a, it is transferred in centrifuge tube, >=8000g centrifugation 1.5
~3min precipitates microballoon；
C. supernatant is removed, dH is added280~120 μ L of O vibrates 15~25s with vortex oscillator and microballoon, >=8000g is resuspended
It is centrifuged 1.5~3min, precipitates carboxyl microballoon；Supernatant is removed, 80~120mmol/L, the sodium dihydrogen phosphate of pH value 6~6.5 is added
60~100 μ L of salting liquid vibrates 15~25s with vortex oscillator, the carboxyl microballoon of washing is resuspended；
D. 8~12 μ L of N hydroxy thiosuccinimide of 40~60mg/ml is added, is lightly vibrated with vortex oscillator；
E. 8~12 μ L of 1- ethyl -3 [3- (dimethylamino) propyl] carbodiimide of 40~60mg/ml is added, is shaken with whirlpool
Device is swung gently to vibrate；
F. it is incubated at room temperature 15~25min, is gently shaken every 8~12min with vortex oscillator, >=8000g centrifugation 1.5~
3min precipitates the carboxyl microballoon of activation；
G. supernatant is removed, 40~60mmol/L, 2- (N- morpholine) ethanesulfonic acid of pH value 4.8~5.2, vortex oscillation is added
Device vibrates 15~25s, and the carboxyl microballoon of activation is resuspended, and >=8000g is centrifuged 1.5~3min, the carboxyl microballoon after washing of precipitate；Weight
The multiple step 2~3 time are washed 2~3 times with the MES of 40~60mmol/L, pH value 4.8~5.2,40~60mmol/L, pH are added
The MES of value 4.8~5.2 vibrates 15~25s with vortex oscillator, the IL- of 40~60 μ g is separately added into the microballoon of mixing
1RA captures antibody, is settled to 400~600 μ L with 40~60mmol/L, the MES of pH value 4.8~5.2, mixed with vortex oscillator
It is even；It is placed in 1.5~3h of incubation on shaking table in room temperature, >=8000g is centrifuged 1.5~3min, precipitates the microballoon being coupled；
H. supernatant is removed, 200~400 μ L of PBS-TBN is added, vortex oscillator vibrates 25~35s；It is placed in and shakes in room temperature
25~35min is incubated on bed, >=8000g is centrifuged 1.5~3min, precipitates the microballoon being coupled；
I. supernatant is removed, 0.8~1.2ml of PBS-TBN is added, vortex oscillator vibrates 25~35s, >=8000g centrifugation
1.5~3min precipitates the microballoon being coupled；Step l~2 time are repeated, are washed 2~3 times with PBS-TBN；
J. 0.8~1.2ml of PBS-TBN is added, coupling is resuspended well and the microballoon by washing is anti-to get IL-1RA capture
The couplet of body and microballoon；
K. the quantity of microballoon is counted with cell counter, concentration is 2~3 × 105A/ml；The microballoon being coupled is placed in 2
~6 DEG C are kept in dark place.
The present invention connects biotin on IL-1RA detection antibody, obtains the IL-1RA detection antibody of biotin labeling.?
In the present invention, the method for the connection preferably includes following steps:
1. the IL-1RA of 0.8~1.2mg is detected 0.08~0.12mol/L of antibody, the bicarbonate of pH value 7.8~8.2 by
Sodium buffer is diluted to 0.8~1.2mg/ml, and final volume is 0.8~1.2ml；
2. 0.08~0.12mol/L of interaction, the sodium bicarbonate buffer liquid of pH value 7.8~8.2 sufficiently dialyse to protein；
3. with 0.8~1.2ml dmso solution N- hydroxyl succinimide activated biotin, 0.8~1.2mg；
4. the NHSB solution 100~150 of 0.8~1.2g/L is added into the IL-1RA of 0.8~1.2ml detection antibody-solutions
μL；It is continuously stirred at room temperature, keeps the temperature 2~4h；
5. the NH of 0.8~1.2mol/L is added49~10 μ L of Cl solution, is stirred at room temperature 8~12min, at 2~6 DEG C
It sufficiently dialyses to PBS, to remove free biotin；By the molecular sieve column of 0.8~1.2ml on sample, slowly eluted with PBS,
0.8~1.2ml/ pipe is collected, protein elutes between 1-3ml；Sample be added final concentration of 0.4~0.6g/L Sodium azide and
The BSA of 0.8~1.2g/L；2~6 DEG C will be placed in conjunction with product to be kept in dark place.
The present invention is not particularly limited the source of the phycoerythrin of the marked by streptavidin, this field conventional commercial
Product is prepared using conventional method in that art.
The present invention provides a kind of methods for blocking curative effect judgement for immunologic test point, preferably include following steps:
(1) before carrying out immunologic test point blocking treatment, IL-1RA marker in the blood serum sample of subject is measured
Content；
(2) after carrying out immunologic test point blocking treatment, IL-1RA marker in the blood serum sample of subject is measured
Content；
(3) measured value in step (1) is compared with the measured value in (2), calculates IL-1RA content in serum
Changing value, changing value is defined as: before (before treatment post-treatment)/treatment.By the size of the changing value, immunologic test point is hindered
Disconnected curative effect is judged.
In the present invention, the immunologic test point Blocking therapy is based on blocking PD-1/PD-L1 or CTLA4 immunologic test
The treatment method of point access, mainly using the specific antibodies medicine of PD-1, the specific antibodies medicine of PD-L1 or CTLA4
Specific antibodies medicine.Method provided by the invention can apply to the cancer patient of gastroenteric tumor, including the cancer of the esophagus, gastric cancer,
Colorectal cancer, liver cancer, cancer of pancreas, cholangiocarcinoma patients.In the cancer of the esophagus, the IL-1RA changing value obtained by abovementioned steps is less than-
14.38% tumor patient is predicted effective to immunologic test point blocking treatment；In colorectal cancer, obtained by abovementioned steps
Tumor patient of the IL-1RA changing value less than 0 is predicted effective to immunologic test point blocking treatment.
Below with reference to embodiment to provided by the invention a kind of for predicting the Serology biological of cancer immunotherapy effect
Marker and its application in kit of the preparation for predicting cancer immunotherapy effect are described in detail, but not
They can be interpreted as limiting the scope of the present invention.
Embodiment 1
The preparation of liquid phase chip reagent box for IL-1RA biological marker analyte detection
1, kit forms
(1) 3-plex is coated with microballoon: containing the coding microball for being coated with IL-1RA capture antibody；
(2) 3-plex biotin labeling detects antibody: detecting antibody with the IL-1RA of biotin labeling；
(3) streptavidin phycoerythrin.
Wherein, the IL-1RA captures antibody, and clone number is 998A2A2；The IL-1RA detects antibody, clone number
For A71B6D11.
2, the preparation method of kit
The following steps are included:
(1) respective capture antibody is coated with corresponding microballoon
A. it takes carboxyl microballoon vortex oscillator to vibrate microsphere suspensions 20s, is uniformly mixed microballoon；
B. carboxyl microballoon 1 × 10 is taken6It is a, it is transferred in centrifuge tube, >=8000g is centrifuged 2min, precipitates microballoon；
C. supernatant is removed, dH is added2Microballoon, >=8000g centrifugation is resuspended with vortex oscillator oscillation 20s in 100 μ L of O
2min precipitates carboxyl microballoon；Supernatant is removed, 100mmol/L is added, the 80 μ L of biphosphate sodium salt solution of pH value 6.2 uses whirlpool
Oscillator vibrates 20s, and the carboxyl microballoon of washing is resuspended；
D. the 10 μ L of N hydroxy thiosuccinimide of 50mg/ml is added, is lightly vibrated with vortex oscillator；
E. 1- ethyl -3 [3- (dimethylamino) propyl] carbodiimide 10 μ L of 50mg/ml is added, it is light with vortex oscillator
Light oscillation；
F. it is incubated at room temperature 20min, is gently shaken every 10min with vortex oscillator, >=8000g is centrifuged 2min, precipitates activation
Carboxyl microballoon；
G. supernatant is removed, 50mmol/L, 2- (N- morpholine) ethanesulfonic acid of pH value 5.0, vortex oscillator oscillation is added
20s, is resuspended the carboxyl microballoon of activation, and >=8000g is centrifuged 2min, the carboxyl microballoon after washing of precipitate；The step 2 time is repeated, is used
The MES of 50mmol/L, pH value 5.0 are washed 2 times, and 50mmol/L, the MES of pH value 5.0 is added, and vibrate 20s with vortex oscillator,
50 μ g IL-1RA capture antibody is added in the microballoon of mixing, is settled to 500 μ L with 50mmol/L, the MES of pH value 5.0, uses whirlpool
Oscillator mixes；It is placed on shaking table in room temperature and is incubated for 2h, >=8000g is centrifuged 2min, precipitates the microballoon being coupled；
H. supernatant is removed, 300 μ L of PBS-TBN is added, vortex oscillator vibrates 30s；It is placed on shaking table and is incubated in room temperature
30min, >=8000g are centrifuged 2min, precipitate the microballoon being coupled；
I. supernatant is removed, PBS-TBN 1ml is added, vortex oscillator vibrates 30s, and >=8000g is centrifuged 2min, precipitating coupling
Good microballoon；It repeats the step l times, is washed 2 times with PBS-TBN；
J. PBS-TBN 1ml is added, the good and microballoon by washing of resuspension coupling is to get IL-1RA capture antibody and microballoon
Couplet；
K. the quantity of microballoon is counted with cell counter, concentration is 2.5 × 105A/ml；The microballoon being coupled is placed in 4
It DEG C is kept in dark place；
(2) IL-1RA detects the biotinylation of antibody
L. the IL-1RA of 1mg is detected into antibody 0.1mol/L, the sodium bicarbonate buffer liquid of pH value 8.0 is diluted to 1mg/
Ml, final volume 1ml；
M. interaction 0.1mol/L, the sodium bicarbonate buffer liquid of pH value 8.0 sufficiently dialyse to protein；
N. 1ml dmso solution N- hydroxyl succinimide activated biotin 1mg is used；
O. the 120 μ L of NHSB solution of 1g/L is added into the IL-1RA of 1ml detection antibody-solutions；It persistently stirs at room temperature
It mixes, keeps the temperature 2-4h；
P. 1mol/LNH is added49.6 μ L of Cl solution, is stirred at room temperature 10min, sufficiently dialyses at 4 DEG C to PBS, with
Remove free biotin；By the molecular sieve column of 1ml on sample, slowly eluted with PBS, collect 1ml/ pipe, protein 1~
It is eluted between 3ml；The Sodium azide and 1.0g/LBSA of final concentration of 0.5g/L is added in sample；4 DEG C will be placed in conjunction with product be protected from light guarantor
It deposits.
Embodiment 2
Application of the IL-1RA liquid phase chip reagent box in prediction cancer immunotherapy effect
1, experiment purpose
Prove that the content of IL-1RA is significantly reduced in the effective tumor patient content of immunologic test point blocking treatment.
2, experimental subjects
1) it under the premise of condition is included in informed consent and satisfaction, selects to record personal essential information into a group case.Integration
Tumor patient queue from 51 advanced gastrointestinal carcinoma patients of tumour hospital, Peking University (including 15 Cases of Gastric Carcinoma, oesophagus
Cancer 15, colorectal cancer 15, liver cancer 4, cancer of pancreas 6 and cholangiocarcinoma 1).
2) the basic clinical information of patient is recorded: including PD-L1 expression quantity, microsatellite instability, recurrence-free survival rate etc..
3) serum before and after tumor patient immunization therapy is extracted, sample in -80 DEG C of refrigerators as saving.
3, reagent prepares
Using the kit prepared in embodiment 1.
(1) Beads: by required Beads (microballoon) ultrasound 30 seconds, vortex 1min, 60 μ L are then respectively taken out, Mixing is added
Bottle, residual volume supply 3ml with Bead Diluent, mix well, and 2~8 DEG C are stored one month.
(2) Quality Control: dissolving control 1 and 2 with 250 μ L distilled water respectively, reverse repeatedly to make it sufficiently mixed
Even, then static 5-10min is moved into two test tubes respectively, and -20 DEG C are stored one month.
(3) Standard: dissolving Standard with 250 μ L distilled water, reverse repeatedly to mix well it, static 5~
10min is then moved into test tube, is labeled as S6.Then 5 test tubes are separately taken, S5, S4, S3, S2, S1, Mei Geguan are respectively labeled as
In plus 200 μ LAssay buffers, finally taken out from S6 50 μ L carry out gradient dilution, -20 DEG C store one month.
(4) Wash Buffer: by 10 × WB place room temperature, make wherein salinity sufficiently dissolve, 30ml WB+270ml distillation
Water is made into 1 × (1 times), and 4 DEG C save one month.
(5) Serum Matrix: 1ml distilled water, which is added, into SM dissolves it sufficiently, and static 10min then moves into examination
Guan Li, -20 DEG C are stored one month.
4, experiment flow:
1. 200 μ LWash Buffer are added in every hole into 96 orifice plates, room temperature shakes rinse in ten minutes, then directly outwells, fill
Divide and dries.
2. being separately added into 25 μ L；
@SerumMatrix to Background, Standard and Control；
@AssayBuffer is to sample well；
@AssayBuffer to Background；
@Standard and Control is to respective position；
@sample is to counter sample hole；
For@Beads to each hole, 4 DEG C are protected from light overnight shaking incubation.
3., board-washing machine-wash 2 times.
4., every hole add 25 μ L detect antibody, room temperature, which is protected from light, shakes 1h.
5., every hole add 25 μ L SAPE, room temperature, which is protected from light, shakes 30min.
6., board-washing machine-wash 2 times, last every hole adds machine (Luminex system) on 150 μ L sheath fluids to detect.
5, experimental result:
IL-1RA changes of contents is imitated in treatment before and after liquid phase chip reagent box detects patients with gastrointestinal carcinoma immunization therapy
There are significant difference in the different tumor patient of fruit, concrete outcome is as shown in Figure 2.Wherein, Fig. 2-A is gastroenteric tumor (packet
Include the cancer of the esophagus, gastric cancer, colorectal cancer, liver cancer, cancer of pancreas, cholangiocarcinoma) patient 51；Fig. 2-B is patient with esophageal carcinoma 15；Fig. 2-
C is colorectal cancer patients 15.The conspicuousness P value that sided t is examined is shown in diagram.Utilize IL-1RA biology before and after immunotherapy of patients
The Receiver operating curve's (ROC curve) for the validity prediction that the content difference of marker treats patients with gastrointestinal carcinoma
See Fig. 3, wherein Fig. 3-A is gastroenteric tumor (including the cancer of the esophagus, gastric cancer, colorectal cancer, liver cancer, cancer of pancreas, cholangiocarcinoma) patient
51；Fig. 3-B is patient with esophageal carcinoma 15；Fig. 3-C is colorectal cancer patients 15.Area under the curve, confidence interval and P value are shown in
Diagram.
Variation [the changing value definition: (treatment post-treatment of IL-1RA biomarker in Tumor Patient Before and After Treatment serum
Before) before/treatment] figure is shown in the recurrence-free survival rate effect being classified and predicted after the cancer of the esophagus and colorectal cancer patients treatment
4.Wherein, Fig. 4-A indicates the PD-L1 expression in tumor tissues, the IL- in microsatellite instability and pretherapy and post-treatment serum
Distribution situation of the 1RA changing value in patient with esophageal carcinoma；The preceding IL-1RA content of relatively treatment is substantially reduced after Fig. 4-B indicates treatment
The cancer of the esophagus of (changing value < -14.38%), recurrence-free survival rate is considerably longer than IL- after receiving immunologic test point blocking treatment
1RA changing value is higher than the patient of above-mentioned threshold value (changing value >=-14.38%)；Fig. 4-C indicates the PD-L1 expression in tumor tissues,
Distribution situation of the IL-1RA changing value in colorectal cancer patients in microsatellite instability and pretherapy and post-treatment serum；Fig. 4-
D relatively treats the cancer of the esophagus that preceding IL-1RA content is substantially reduced (changing value≤0) after indicating treatment, blocks receiving immunologic test point
Recurrence-free survival rate is considerably longer than the patient that IL-1RA changing value is higher than above-mentioned threshold value (changing value > 0) after treatment.Statistical check knot
Fruit sees diagram.
The experimental results showed that calculating IL-1RA detection and changing value in serum before and after immunization therapy, it can be achieved that tumour
The Accurate Prediction of immunotherapy of patients effect, predictablity rate is up to 77% or more.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of for predicting the Serology biological marker of cancer immunotherapy effect, which is characterized in that the serum student
Object marker is IL-1RA.
2. biomarker according to claim 1, which is characterized in that the cancer includes gastroenteric tumor.
3. biomarker according to claim 2, which is characterized in that the gastroenteric tumor include the cancer of the esophagus, gastric cancer,
One or more of colorectal cancer, liver cancer, cancer of pancreas and cholangiocarcinoma.
4. Serology biological marker described in claims 1 to 3 any one is in preparation for predicting that cancer immunotherapy is imitated
Application in the kit of fruit.
5. a kind of for predicting the kit of cancer immunotherapy effect, which is characterized in that be coated in the kit
The coding microball of IL-1RA capture antibody, the IL-1RA of biotin labeling detect the algae red egg of antibody and marked by streptavidin
It is white.
6. kit according to claim 5, which is characterized in that the clone number of IL-1RA capture antibody is
998A2A2。
7. kit according to claim 5, which is characterized in that the clone number of IL-1RA detection antibody is
A71B6D11。
8. kit according to claim 5, which is characterized in that the coding microball includes carboxyl microballoon.
9. kit according to claim 5, which is characterized in that the biotin includes the activation life of N- carboxylic succinimide
Object element.
10. the preparation method of kit described in claim 5~9 any one, which comprises the steps of:
(1) IL-1RA capture antibody and coding microball are coupled, obtain the coding microball for being coated with IL-1RA capture antibody；
(2) biotin is connected on IL-1RA detection antibody, obtains the IL-1RA detection antibody of biotin labeling；
The step (1) and (2) are limited without time sequencing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910109792.9A CN109655624A (en) | 2019-02-11 | 2019-02-11 | A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910109792.9A CN109655624A (en) | 2019-02-11 | 2019-02-11 | A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109655624A true CN109655624A (en) | 2019-04-19 |
Family
ID=66121306
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910109792.9A Pending CN109655624A (en) | 2019-02-11 | 2019-02-11 | A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109655624A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110456042A (en) * | 2019-08-09 | 2019-11-15 | 臻悦生物科技江苏有限公司 | Application, kit or chip and preparation method thereof of the Serology biological marker in reagent preparation box and/or chip |
| CN111562362A (en) * | 2020-04-02 | 2020-08-21 | 臻悦生物科技江苏有限公司 | Marker for predicting three-negative breast cancer immune combined anti-angiogenesis treatment effect and application and kit thereof |
| CN113358872A (en) * | 2021-06-03 | 2021-09-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Marker group and system for evaluating curative effect of tumor immunotherapy |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050153329A1 (en) * | 2002-05-27 | 2005-07-14 | Leif Hakansson | Method for determining immune system affecting compounds |
| CN101526535A (en) * | 2009-04-14 | 2009-09-09 | 河南省豫康生物工程技术有限公司 | Liquid phase chip for joint detection of multiple tumor markers and preparation method thereof |
| CN102539745A (en) * | 2010-12-31 | 2012-07-04 | 中国人民解放军第三〇九医院 | Early diagnosis and early warning kit for transplanted kidney rejection reaction and detection method |
| CN102854305A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Prewarning kit for transplant kidney rejection and use method thereof |
| US20130330325A1 (en) * | 2010-09-24 | 2013-12-12 | Niels Grabe | Means and methods for the prediction of treatment response of a cancer patient |
| US20150301058A1 (en) * | 2012-11-26 | 2015-10-22 | Caris Science, Inc. | Biomarker compositions and methods |
| CN105785005A (en) * | 2016-04-13 | 2016-07-20 | 杭州华得森生物技术有限公司 | Circulating tumor cell detection kit and application thereof |
| CN107988373A (en) * | 2018-01-10 | 2018-05-04 | 上海交通大学医学院附属仁济医院 | For predicting the biomarker, kit and application of cancer immunotherapy effect |
-
2019
- 2019-02-11 CN CN201910109792.9A patent/CN109655624A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050153329A1 (en) * | 2002-05-27 | 2005-07-14 | Leif Hakansson | Method for determining immune system affecting compounds |
| CN101526535A (en) * | 2009-04-14 | 2009-09-09 | 河南省豫康生物工程技术有限公司 | Liquid phase chip for joint detection of multiple tumor markers and preparation method thereof |
| US20130330325A1 (en) * | 2010-09-24 | 2013-12-12 | Niels Grabe | Means and methods for the prediction of treatment response of a cancer patient |
| CN102539745A (en) * | 2010-12-31 | 2012-07-04 | 中国人民解放军第三〇九医院 | Early diagnosis and early warning kit for transplanted kidney rejection reaction and detection method |
| CN102854305A (en) * | 2011-12-23 | 2013-01-02 | 中国人民解放军第三〇九医院 | Prewarning kit for transplant kidney rejection and use method thereof |
| US20150301058A1 (en) * | 2012-11-26 | 2015-10-22 | Caris Science, Inc. | Biomarker compositions and methods |
| CN105785005A (en) * | 2016-04-13 | 2016-07-20 | 杭州华得森生物技术有限公司 | Circulating tumor cell detection kit and application thereof |
| CN107988373A (en) * | 2018-01-10 | 2018-05-04 | 上海交通大学医学院附属仁济医院 | For predicting the biomarker, kit and application of cancer immunotherapy effect |
Non-Patent Citations (5)
| Title |
|---|
| TERESA URBANOWSKA 等: "Protein microarray platform for the multiplex analysis of biomarkers in human sera", 《JOURNAL OF IMMUNOLOGICAL METHODS》 * |
| 无: "IL1RA Monoclonal Antibody (998A2A2)", 《THERMO FISHER》 * |
| 无: "IL1RA Monoclonal Antibody (A71B 6D11), Biotin", 《THERMO FISHER》 * |
| 杨帆等: "液相芯片技术在胃癌多靶标基因联合检测中的应用", 《细胞与分子免疫学杂志》 * |
| 高基民等主编: "《分子诊断学实验指导》", 28 February 2010 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110456042A (en) * | 2019-08-09 | 2019-11-15 | 臻悦生物科技江苏有限公司 | Application, kit or chip and preparation method thereof of the Serology biological marker in reagent preparation box and/or chip |
| CN111562362A (en) * | 2020-04-02 | 2020-08-21 | 臻悦生物科技江苏有限公司 | Marker for predicting three-negative breast cancer immune combined anti-angiogenesis treatment effect and application and kit thereof |
| CN111562362B (en) * | 2020-04-02 | 2022-05-20 | 臻悦生物科技江苏有限公司 | Marker for predicting three-negative breast cancer immune combined anti-angiogenesis treatment effect and application and kit thereof |
| CN113358872A (en) * | 2021-06-03 | 2021-09-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Marker group and system for evaluating curative effect of tumor immunotherapy |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109655624A (en) | A kind of preparation method of marker and its application for predicting cancer immunotherapy effect, kit and kit | |
| Inverso et al. | A spatial vascular transcriptomic, proteomic, and phosphoproteomic atlas unveils an angiocrine Tie–Wnt signaling axis in the liver | |
| US8198024B2 (en) | Gene expression markers for colorectal cancer prognosis | |
| ES2445892T3 (en) | Biomarkers for anti-TNF treatment in ulcerative colitis and related disorders | |
| CN102165315B (en) | Markers and methods for assessing and treating ulcerative colitis and related disorders using a 20 gene panel | |
| CN110456042A (en) | Application, kit or chip and preparation method thereof of the Serology biological marker in reagent preparation box and/or chip | |
| CN101416058A (en) | Wnt proteins and detection and treatment of cancer | |
| JP2016536002A (en) | System, device, and method for anti-TL1A therapy | |
| AU2018317865A1 (en) | Polymorphic form of TG02 | |
| JP2021535100A (en) | Methods and Treatments for Modulating M2 Macrophage Polarization | |
| CN105985962A (en) | Aptamer specifically targeting to inflammatory synovial cells of rheumatoid arthritis (RA) and applications of aptamer | |
| US11466324B2 (en) | Lipocalin 2 as a biomarker for IL-17 inhibitor therapy efficacy | |
| KR20140080504A (en) | Screening methods and uses thereof | |
| US20210293814A1 (en) | Systems and methods for spectral imaging characterization of macrophages for use in personalization of targeted therapies to prevent fibrosis development in patients with chronic liver disease | |
| CN106950384A (en) | Rectal neoplasm mark and its application, kit | |
| Philip et al. | AT cell repertoire timestamp is at the core of responsiveness to CTLA-4 blockade | |
| JP6095984B2 (en) | Prognostic markers for cryoglobulinemia vasculitis and B cell malignancies in HCV infected patients | |
| CN109081866A (en) | T cell subgroup and its characterizing gene in cancer | |
| CN111562362B (en) | Marker for predicting three-negative breast cancer immune combined anti-angiogenesis treatment effect and application and kit thereof | |
| CN112255410B (en) | Marker for predicting 2019 coronavirus immune checkpoint storm, application and kit thereof | |
| CN111220811A (en) | Screening method of TrpRS inhibitor | |
| CN112162097B (en) | GDF1 as biomarker for evaluating treatment effect of PD-1 monoclonal antibody | |
| EP0871883A1 (en) | Compounds and methods related to parathyroid hormone-like protein | |
| JP2006520588A (en) | Therapeutic, preventive, and diagnostic agents | |
| CN108753959B (en) | SNP marker located in DISC1FP1 gene and related to radioactive brain injury caused by radiotherapy and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: Room 401, block D, Xidong Chuang Rong building, 88 Danshan Road, anzhen street, Wuxi City, Jiangsu Province Applicant after: Wuxi Zhenhe Biotechnology Co.,Ltd. Address before: 100000 No. 9 garden north road, Beijing, building 9, floor 35, 908, Haidian District Applicant before: GENECAST (BEIJING) TECHNOLOGY Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 1310-1318, block C, Xidong chuangfong building, 88 Danshan Road, anzhen street, Xishan District, Wuxi City, Jiangsu Province, 214104 Applicant after: Wuxi Zhenhe Biotechnology Co.,Ltd. Address before: Room 401, block D, Xidong chuangfong building, 88 Danshan Road, anzhen street, Shanshan District, Wuxi City, Jiangsu Province, 214104 Applicant before: Wuxi Zhenhe Biotechnology Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 214101 1310-1318, block C, Xidong chuangrong building, No. 88, Danshan Road, anzhen street, Xishan District, Wuxi City, Jiangsu Province Applicant after: Wuxi Zhenhe Biotechnology Co.,Ltd. Address before: 1310-1318, block C, Xidong chuangfong building, 88 Danshan Road, anzhen street, Xishan District, Wuxi City, Jiangsu Province, 214104 Applicant before: Wuxi Zhenhe Biotechnology Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190419 |
|
| RJ01 | Rejection of invention patent application after publication |