CN104076151B - Kit for early diagnosis of glioma - Google Patents

Abstract

The invention belongs to the technical field of biology, and relates to a kit for tumor diagnosis, in particular to a kit for early diagnosis of glioma. The kit comprises a mouse anti-human B7-H4 biotin labeling monoclonal antibody and streptavidin-peroxidase working liquid, a collected cerebrospinal fluid sample is detected, and corresponding diagnosis can be made by detecting B7-H4 molecules in the cerebrospinal fluid. The kit can be used for quantitatively detecting the B7-H4 protein expression quantity in the cerebrospinal fluid, so that the glioma infection risk can be predicted, people at high risk can be screened, the early diagnosis of glioma can be realized, and glioma and other intracranial space occupying diseases can be distinguished. The kit has the characteristics that the sensitivity is 70.8 percent, the specificity is 100 percent, the sensitivity for distinguishing low-grade and top-grade glioma is 87.5 percent, and the specificity for distinguishing low-grade and top-grade glioma is 75.0 percent. The kit is free of cross reaction with other cell factors, so that the powerful technical support is provided for the accurate diagnosis and prevention of glioma.

Description

A kind of test kit for glioma early diagnosiss

Technical field

The invention belongs to biological technical field, is related to tumor diagnosis kit, and in particular to a kind of new to can be used for glue The test kit of matter tumor early diagnosiss, the test kit not only can be entered by the B7-H4 molecules in detection cerebrospinal fluid to high-risk group Row examination, and neoplasm staging can be instructed, it is to find sensitive, special glioma cerebrospinal fluid mark to provide the foundation.

Background technology

Research shows, in Primary intracranial tumor, cerebral glioma sickness rate highest, and close 40.5%.Glioma is accounted for The 20% of virgin tumor, is also the dead primary cause of disease of solid tumor infant.According to investigations, glioma onset peak has 30-40 year Life cycle is short, relapse rate and the features such as high mortality rate.Especially glioblastoma multiforme, its 2 years survival rates are 27.2%, life in 5 years The rate of depositing is only 9.8%, newest although Comprehensive Treatment (including operation, radiotherapy, chemotherapy) in recent years has made great progress Research shows its median survival interval still less than 20 months.Therefore, how examination and early stage are carried out to glioma high-risk group Diagnosis, to start to treat in time, to extend patient survival, becomes the problem of increasing scientist and doctor's concern.

Glioma clinical manifestation mainly includes the symptom and sign such as increased intracranial pressure and neurological deficit, and specificity is not high, Therefore, the preoperative diagnosis of current glioma rely primarily on Imaging Technology, with MRI it is unenhanced plus strengthen based on, supplemented by CT, additionally, PET, SPECT and MRI specific function inspection such as MRS, PWI, DWI, DTI etc. can also be used for Differential Diagnosiss, preoperative evaluation, recurrence inspection Survey, but these imaging diagnosises, according to the stage still in empirical standard, still can not meet carries out high-risk people to cerebral glioma The demand that group's examination, early diagnosiss and other intracranial space-occupying lesions differentiate.

B7-H4 as B7 families in new negativity costimulatory moleculeses, its effect in tumour immunity enjoys in recent years Attract attention.Research shows that the abundant activation of T lymphocytes is the key of cerebral tumor immunity.The activation of T cell at least needs two letters Number：First signal by T cell surface antigen-specific receptor and antigen-presenting cell (APC) or the MHC of tumor cell surface Antigenic peptide complexes are combined and provided；Secondary signal is then by the costimulatory moleculeses (costimulatory molecules) on APC Produce to the corresponding receptor binding on T cell surface, the signal can affect activation, propagation and the cytokine secretion of T cell.Pierce altogether Sharp molecule can be divided into positivity and negativity, keep being balanced for maintenance body immune system function just in positive negative costimulatory signal Often, avoid the unnecessary activation of T cell or suppress significant, and tumor cell is often through low expression positivity costimulatory moleculeses Or height expresses negativity costimulatory moleculeses to escape from the immune surveillance of body.B7 families contain many important collaborations stimulations point Son, such as B7-1, B7-2, B7-H1, can promote or suppress T cell propagation and cytokine to produce, and B cell activation, antibody are produced Life also plays important regulative.DM Kuang etc. have found that B7-H1 positive macrophages can suppress tumor special in hepatocarcinoma substrate Specific T cell immunity, and the prognosis of the gather density of this group of macrophage in hepatocarcinoma and hepatocarcinoma patient is in negative correlation.For Newly discovered B7-H4, has studied and has confirmed that brain Tumor Stem Cells (brain tumor stem cells) can pass through B7- There is immunologic escape in H1/B7-H4 approach.

With going deep into for studying B7-H4, there are some scholars to start to be conceived to the relation of B7-H4 and tumor prognosis. Arigami etc. find content and the Patients with Gastric Cancer of B7-H4 in tumor tissues by stages, 5 years survival rates it is significantly correlated, it is possible to make For the independent influencing factor of gastric cancer prognosis, additionally, they are investigated the existence point of peripheral blood lymphocyte B7-H4 expression Analysis, and obtained similar result.Chen etc. has carried out the SABC of squamous cell carcinoma of esophagus, and Jing analyses also demonstrate that The tumour patient of the high expression of B7-H4 is presented worse prognosis.Renal carcinoma, melanoma field also demonstrate B7-H4 in tumor in succession Importance in terms of prognosis.Wherein, most it is noticeable be Irish etc. research, they have found the expression of B7-H4 in ovarian cancer Independently of CA125, if by serum B7-H4 be individually used for diagnosis, specificity be 97% when sensitivity up to 45%, and if will B7-H4 combines with CA125, and sensitivity can be improved to 65% and be better than traditional CA125 diagnostic methods.These all point out B7-H4 to incite somebody to action Can be used as a kind of novel tumor markers.

ELISA (enzyme-linked immunosorbent assay) technology is that the antigen or antibody of solubility are adsorbed onto into the solid phases such as polystyrene On carrier, the qualitative and quantitative approach of immunoreation is carried out.Its ultimate principle is to prepare enzyme-labelled antigen or antibody first, even if anti- Former or antibody is connected with certain enzyme, has both retained its immunocompetence, and the activity of enzyme is retained again, then by inspection specimen, (measure is wherein Antibody or antigen) and enzyme-labelled antigen or antibody react with the antigen or antibody of surface of solid phase carriers by different steps, wash Wash the antigen antibody complex formed on purification solid phase carrier so that combine enzyme amount and examined object in specimen on solid phase carrier The amount of matter is eventually adding the substrate of enzyme reaction, the amount and specimen of the color products that substrate is become by enzyme catalysiss into certain ratio The amount of middle tested substance is directly related, therefore can carry out qualitative or quantitative analysis according to the depth of color reaction.Therefore, in view of ELISA method have the advantages that simple to operate, sensitivity it is high, can be quantitative, applicant of the present invention intend by with ELISA detect brain ridge Liquid B7-H4 contents, set up a kind of method that can carry out early screening, diagnosis, Differential Diagnosiss to patients with gliomas, and this is to glioma Prevention, treatment be of great importance.

The content of the invention

It is an object of the invention to provide new glioma tumor mark B7-H4, the present invention is by studying negativity costimulation Expressions of the molecule B7-H4 in gliomatosis human cerebrospinal fluid, it is proposed that B7-H4 can be used as glioma tumor mark；

It is a further object of the present invention to provide a kind of test kit for glioma early diagnosiss, in the present invention, by B7-H4 Glioma tumor mark is used for diagnosis of glioma, examination and assessment prognosis, can solve current glioma early diagnosiss difficulty, mirror Do not diagnose and excessively rely on the problem of pathology and be applied to correlated therapeutic regimens and prognosis evaluation.

Present invention research shows that human brain tumour stem cell can express B7-H4, the correlation to Mus GL261 glioma cell lines Experiment discloses the higher B7-H4 of the CD133 positive glioma stem cells tumor cells expression negative compared with CD133, in vitro substantially Suppress the immunologic function of T cell, promote the growth of Xenografts in nude mice, research also to show in vivo, CD11b positive little glue Cell plastid/macrophage also expresses B7-H4.

Further, the present invention has carried out the clinical research of B7-H4, and by western blot High Grade Gliomas phase is found More B7-H4 protein are expressed than Low grade glioma, subsequent qRT-PCR also confirms above-mentioned knot from mRNA level in-site Really.Further, in order to more intuitively show expressions of the B7-H4 in tumor tissues, in the present invention, to normal cerebral tissue And the glioma specimen of different stage implements SABC and immunofluorescence dyeing, B7-H4 is demonstrated with tumor rank Increase the expression phenomenon that increases and hardly express in the normal tissue, it is often more important that, according to B7-H4 staining conditions to 64 Example glioblastoma multiforme (WHO IV levels) patient carries out after survival analysises, as a result showing that tumor tissues height expresses the patient of B7-H4 With relatively bad prognosis, B7-H4 is pointed out perhaps to can be used in instructing the prognosis of glioma patient.

In view of microglia/macrophage is the central immune cells in cerebral tissue, in the present invention, purification brain is sorted This kind of cell in tissue and peripheral blood, shows through flow cytometry, and the B7-H4 in High Grade Gliomas tissue is positive Property microglia/macrophage is significantly raised.

The content that the present invention passes through B7-H4 in detection normal person, different stage patients with gliomas cerebrospinal fluid, using statistics Analysis discloses the dependency of B7-H4 and glioma, be glioma clinical osology diagnose propose B7-H4 can be used as glioma Tumor markerses.

The invention discloses a kind of diagnosis of glioma test kit and its preparation method and application, the test kit can accurately, The B7-H4 in cerebrospinal fluid is rapidly detected, and then timely auxiliary diagnosis can be made to each phase glioma, faced with higher Bed using value.

It is for glioma early diagnosiss and the test kit of Differential Diagnosiss including the anti-human B7-H4 monoclonal antibodies of Mus, peppery in the present invention Root peroxidase labelling two is anti-, common reagent Tween-20, chromogenic substrate ABTS, ELISA Plate etc..The test kit can be quantitative B7-H4 expressing quantities in detection cerebrospinal fluid, so as to predict the risk of glioma, examination high-risk group, early diagnosiss glue Matter tumor patient, differentiates glioma and other intracranial space-occupying lesions.Its sensitivity is high, can detect the B7-H4 of 30pg/ml, specifically Property it is high, other cytokines do not have cross reaction with people, and the Accurate Diagnosis and preventing and treating for glioma provide very strong skill Art basis.

Certain dependency is had with the tumor rank of patients with gliomas based on contents of the described B7-H4 in cerebrospinal fluid, Therefore, test kit of the invention is additionally operable to assess the prognosis of glioma.

Description of the drawings

Fig. 1：Western blot detections normal cerebral tissue, different stage glioma B7-H4 expression.

Fig. 2：Immunohistochemical staining detection normal cerebral tissue, different stage glioma B7-H4 expression.

Fig. 3：B7-H4 positive microglial cells ratios in Flow cytometry normal cerebral tissue, different stage glioma.

Fig. 4：B7-H4 concentration in ELISA detection glioma patients, non-tumour patient cerebrospinal fluid.

Fig. 5：The present invention is used to diagnose the ROC curve of glioma.

Specific embodiment

The present invention does further explaination explanation with respective drawings in conjunction with the embodiments, and following examples are for illustration purposes only, It is not used in the restriction scope of the invention.

Embodiment 1,

First, Western blot detections normal cerebral tissue, different stage glioma B7-H4 expression：

It is prepared by protein example：Cerebral tissue adds homogenate buffer, machinery or ultrasound wave room temperature homogenate 0.5-1min after shredding. Then 4 DEG C, 12,000g centrifugation 15min.Supernatant is taken as sample；

Electrophoresis：SDS-PAGE running gels are prepared, sample-loading buffer is added in sample, after being cooled to room temperature, by protein sample Loading to gel carries out electrophoresis；Transferring film：Electrophoresis cuts adhesive tape to suitable size after terminating, and is balanced with transferring film buffer, 5min × 3 It is secondary.Cut out in advance and an equal amount of filter paper of adhesive tape and NC films, 10min in immersion transferring film buffer；Membrane-transferring device is from bottom to up Put well by the order of carbon anode plate, 24 metafiltration paper, NC films, gel, 24 metafiltration paper, negative electrode carbon plate successively, filter paper, gel, NC films Accurate align, each step goes bubble removing, upper ballast to blot liquid unnecessary on carbon plate；Switch on power, constant current 300mA turns Move 1.5h；After transfer terminates, deenergization takes the film out；

Antibody incubation：Film bar to be measured is cut, with 0.01M PBS film, 5min × 3 time are washed.Confining liquid is added, it is steady to shake, Room temperature 2h.Confining liquid is abandoned, with 0.01M PBS film, 5min × 3 time are washed.Add rabbit-anti people B7-H4 mono- it is anti-(1: 5000, Epitomics), 4 DEG C of placement more than 12h；Negative control, replaces one to resist with 1%BSA, and remaining step is identical with experimental group；Abandon one Anti- and 1%BSA, with 0.01M PBS film, 5min × 4 time are washed respectively.Add the two of horseradish peroxidase to resist, steadily shake It is dynamic, room temperature 2h.Abandon two to resist, with 0.01M PBS film, 5min × 4 time are washed；

Colour developing：Nitrite ion is added, film is prepared in darkroom；

With reference to shown in Fig. 1, it can be seen from shade, High Grade Gliomas expression B7-H4 is more than Low grade glioma, and Normal cerebral tissue hardly expresses；

2nd, immunohistochemical staining detection normal cerebral tissue, different stage glioma B7-H4 expression：

Dewaxing and aquation：Cerebral tissue paraffin section is placed in into 30min in dimethylbenzene I, 20min in dimethylbenzene II, dimethylbenzene 10min in III, soaks 5mins in dehydrated alcohol, and 5min is soaked in 95% ethanol, soaks 5mins in 80% ethanol, and 60% 5min is soaked in ethanol, single steaming in water soaks 5min；

Antigen retrieval：A certain amount of CB buffer is taken in a high-temperature resistant container, in putting microwave oven into, power10 × 2min, is allowed to seethe with excitement, and then section is carefully placed into wherein, then is slowly put into heating in microwave oven, power3 × 5min, most Afterwards, it is placed on room temperature natural cooling 30-40min；

Dyeing：Section is taken out, TBS is rinsed 4-5 time, and drying is cleaned.30ul H2O2 are added, is closed in room temperature wet box 30min, drying is cleaned；The anti-human B7-H4 mono- of Deca Mus resists (1: 50, AbD Serotec) 40ul, and 4 DEG C overnight, and TBST rinses 4-5 Secondary, the anti-20ul of Deca biotinylation two is incubated at room temperature 1h, and TBS is rinsed 4-5 time, Deca enzyme mark Avidin 20ul, incubation at room temperature 30min, TBS are rinsed 4-5 time, are rinsed well rapidly after the coloring of Deca 30ul substrate, the drop hematoxylin of Deca 1,5-10min, dehydration, Mounting, microscopy；

With reference to shown in Fig. 2, brown color dyeing prompting B7-H4 positive cells, contrast understands that High Grade Gliomas B7-H4 is positive Sexual cell is most, and normal structure is only more than negative control；

3rd, B7-H4 positive microglial cells ratios in Flow cytometry normal cerebral tissue, different stage glioma：

Separate microglia：Fresh cerebral tissue specimen (in excision 20h) is taken, is shredded, centrifugation 1500rpm × 5min, abandons supernatant, adds papain (1mg/ml) 4-5ml, 37 DEG C of digestion 30min, increase serum to terminate being centrifuged after digestion 1500rpm × 5min, abandons supernatant, and 1640 culture medium with 10ml are resuspended, crosses 40 μm of filter screens in 15ml centrifuge tubes, centrifugation 1500rpm × 5min, abandons supernatant, and 70%Percoll solution 4ml, 30%Percoll solution 8ml are sequentially added in centrifuge tube, HBSS2ml, is centrifuged 500g × 30min, and from 70% with the interface of 30%Percoll solution cell is drawn, centrifugation after PBS is resuspended 1500rpm × 5min, abandons supernatant；

Immunological magnetic bead sorting：With the resuspended upper step gained cell of the MACS buffer of 80 μ l, the anti-human CD11b of coating is added to resist Supernatant is abandoned in the μ l of immunomagnetic beadses 20 of body, 4 DEG C of 15min, MACS buffer solutions 2 times, centrifugation, and 500 μ l MACS buffer are resuspended thin Born of the same parents, cross post, move post and go out magnetic field, and with the MACS buffer of 1ml post is washed, and collect washing liquid and are positive cell；

Flow cytometry：100 μ l PBS re-suspended cells, plus the anti-human CD11b antibody and 5 μ 1PE of 5 μ l APC combinations With reference to anti-human B7-H4 antibody (1: 5, eBioscience), 4 DEG C of incubation 15min, PBS washes 2 times, and 300 μ l paraformaldehydes are resuspended Cell, upper machine testing；

With reference to shown in Fig. 3, the streaming figure upper right corner be B7-H4 positive microglial cells, High Grade Gliomas, low level colloid Tumor, the B7-H4 positive cell ratios of normal cerebral tissue are reduced successively.

Embodiment 2, the test kit for preparing glioma early diagnosiss

The test kit includes following components：

1st, the hole elisa Plates of solid phase 96；

2nd, the anti-human B7-H4 purified monoclonal antibodies (eBioscience) of Mus；

3rd, the anti-human B7-H4 biotin labelings monoclonal antibody (eBioscience) of Mus；

4th, streptavidin's albumen-peroxidase working solution (Abcam)；

5th, standard substance：B7-H4 albumen (R＆D Systems)；

6th, ABTS substrates：ABTS1mg+0.1M, pH5.0 citrate buffer solution 2ml+3%H2O24ul；

7th, cleaning mixture：0.05%Tween20-PBS；

8th, diluent：0.1%BSA-PBS；

9th, terminate liquid：2M H2SO4.

The detection method of test kit, including：

Standard substance are prepared：The B7-H4 standard substance of different content are dissolved in into diluent, 0.1,0.5,2,10,25 are reached, 50,100ng/ml isoconcentrations；

Coating closing：The anti-human B7-H4 purified monoclonal antibodies of Mus are diluted to 2ug/ml, and ELISA Plate is coated with 100ul, 4 DEG C of mistakes per hole Night.Cleaning mixture is cleaned 6 times, plus diluent 200ul/ holes, 37 DEG C of standing 1h；

Sample-adding：Cleaning mixture cleaning ELISA Plate 6 times, sets respectively blank well, gauge orifice, testing sample hole, and blank well adds sample Diluent 100ul, remaining hole adds respectively standard substance or testing sample 50ul, and light rolling mixes, overlay film in ELISA Plate, 37 DEG C of reaction 2h；

One anti-incubation：Liquid is discarded, is dried, per hole 50ul biotin labelings B7-H4 antibody working solutions (1: 100) is added, 37 Degree Celsius stand 1h；

Enzyme labelled antibody is incubated：Liquid is discarded, is dried, washed 6 times, each 1-2min, 350ul/ holes, dried, added per hole 50ul streptavidins albumen-peroxidase working solution (1: 1000), 37 degrees Celsius of standing 0.5h；

Colour developing：Cleaning mixture cleaning ELISA Plate 6 times, adds 100ul ABTS substrate working solutions (1: 1000) per hole, 37 degrees Celsius Lucifuge colour developing 0.5h；Add 50ul terminate liquids, terminating reaction per hole；

Detection level：With microplate reader in the optical density value (OD values) in each hole of 450nm wavelength measurements, painted according to standard substance OD values Standard curve processed, and then calculate the B7-H4 concentration of specimen to be measured.

Embodiment 3, test kit detection cerebrospinal fluid B7-H4 concentration is used to diagnose sensitivity, the specificity of glioma

Make a collection of specimens：56 patients that 2011-2012 goes to a doctor in Huashan hospital neurosurgery are obtained first (outside 5 brains Wound, 1 meningioma, 2 metastatic tumors, 16 Low grade gliomas, 32 High Grade Gliomas) agreement after, they are carried out Waist is worn, and everyone leaves and takes 5ml cerebrospinal fluid, be stored in -80 DEG C of refrigerators；

Detection B7-H4 concentration：Spinal fluid samples thaw 30-60min in ice bath, and 1500rpm × 5min is centrifuged, and take Clearly, according to the method in embodiment 2, using described test kit, resist through standard substance preparation, coating closing, sample-adding, one and incubate Educate, enzyme labelled antibody incubation, colour developing, microplate reader detection etc. step, measure the B7-H4 concentration of each specimen；

Data statisticss (as shown in Figure 4)：Using GraphPad5.02 softwares；Cerebrospinal fluid B7-H4 concentration mensurations result is adopted Mean value ± SEM represents, can obtain 95.14 ± 16.30ng/ml of non-glioma group, Low grade glioma 123.2 ± 11.25ng/ of group Ml, High Grade Gliomas 291.4 ± 29.51ng/ml of group, glioma 235.3 ± 23.02ng/ml of group；Using t inspections ratio between group Compared with non-glioma group B7-H4 can be obtainedConcentration is substantially less than glioma groupAnd High Grade Gliomas group (P=(P=0.0175) 0.0023), Low grade glioma group B7-H4 concentration is substantially less than High Grade Gliomas group (P=0.0003), rather than glioma group Without significant difference (P=0.1669) between Low grade glioma group；

ROC curve is analyzed：Using SPSS Statistics20 softwares；The ROC curve of diagnosis glioma is drawn (such as Fig. 5 A It is shown), area under curve is 0.854, chooses the maximum point of youden index (sensitivity+specificity -1) as diagnosis glioma Marginal value, as a result shows, sensitive when the standard using cerebrospinal fluid B7-H4 concentration >=133.6170ng/ml as diagnosis glioma Spend for 70.8%, specificity is 100%；Drafting compares Low grade glioma with the ROC curve of High Grade Gliomas (such as Fig. 5 B institutes Show), area under curve is 0.871, chooses the maximum point of youden index as the marginal value of diagnosis glioma, is as a result shown, when It is sensitive during using cerebrospinal fluid B7-H4 concentration 135.1305ng/ml as the standard for differentiating Low grade glioma and High Grade Gliomas Spend for 87.5%, specificity is 75.0%.

The clinical practice of embodiment 4, test kit

The preoperative waist that carries out of a certain doubtful patients with gliomas is worn, and gathers 4ml cerebrospinal fluid, and the method in reference implementation example 2 is adopted The test kit of the present invention, prepares, is coated with closing, sample-adding, an anti-incubation, enzyme labelled antibody incubation, colour developing, microplate reader through standard substance The steps such as detection, measure its B7-H4 concentration；

The absorbance of reference standards, the B7-H4 concentration for drawing the sample is 149.4877ng/ml；According to such as embodiment 3 It is described to the standard specified by 56 gliomas and non-gliomatosis human cerebrospinal fluid B7-H4 concentration mensurations, i.e. cerebrospinal fluid B7-H4 Concentration >=133.6170ng/ml is possible for glioma, and during concentration >=135.1305ng/ml the glioma be possible for it is senior Not, according to the above-mentioned B7-H4 concentration for measuring, the patient is diagnosed for High Grade Gliomas；Jing postoperative pathologicals are as a result, it was confirmed that the patient For glioma WHO IV levels.

Claims (2)

1. cerebrospinal fluid B7-H4 albumen prepare glioma Differential Diagnosiss preparation in purposes；Described B7-H4 albumen is used as colloid Struma tumor markers；Wherein, during cerebrospinal fluid B7-H4 protein concentrations >=133.6170ng/ml, tentative diagnosis is glioma；Brain ridge During liquid B7-H4 protein concentrations >=135.1305ng/ml, tentative diagnosis is High Grade Gliomas.

2. cerebrospinal fluid B7-H4 albumen for prepare glioma early diagnosiss test kit in purposes；Described B7-H4 albumen Used as glioma tumor mark, the test kit detects B7-H4 protein concentrations in sample to be tested cerebrospinal fluid, wherein, B7-H4 contains Amount and cerebrospinal fluid donor are related without there is glioma that there is the glioma rank of dependency, B7-H4 contents and cerebrospinal fluid donor to have Property.