CN104076151A - 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 kit for glioma early diagnosis

Technical field

The invention belongs to biological technical field, relate to tumor diagnosis kit, be specifically related to a kind of novel kit that can be used for glioma early diagnosis, this kit is by detecting the B7-H4 molecule in cerebrospinal fluid, not only can carry out examination to people at highest risk, and can instruct neoplasm staging, for finding sensitive, special glioma cerebrospinal fluid mark, provide the foundation.

Background technology

Studies show that, in Primary intracranial tumor, the glioma incidence of disease is the highest, approaches 40.5%.Glioma accounts for 20% of pediatric tumor, is also the primary cause of disease of solid tumor infant death.According to investigations, glioma onset peak is in 30-40 year, has short, recurrence rate and mortality ratio high life cycle.Especially glioblastoma, its 2 years survival rates are 27.2%, within 5 years, survival rate is only 9.8%, although complex treatment in recent years (comprising operation, radiotherapy, chemotherapy) has obtained very much progress, its median survival interval is still less than 20 months in up-to-date studies show that.Therefore, how can glioma people at highest risk be carried out to examination and early diagnosis, so as in time begin treatment, extend the survival of patients phase, become the problem that increasing scientist and doctor pay close attention to.

Glioma clinical manifestation mainly comprises the symptom and signs such as increased intracranial pressure and neurological deficit, specificity is not high, therefore, the preoperative diagnosis of current glioma mainly relies on Imaging Technology, the MRI of take is unenhanced adds enhancing as main, CT is auxiliary, in addition, PET, SPECT and MRI specific function check as MRS, PWI, DWI, DTI etc. also can be used for antidiastole, preoperative assessment, recurrence detects, but these imaging diagnosises foundations are the stage in empirical standard still, still can not meet glioma is carried out to people at highest risk's examination, early diagnosis, demand with other intracranial space-occupying lesions discriminatings.

B7-H4 is as the novel negativity costimulatory molecules in B7 family, and its effect in tumour immunity gets most of the attention in recent years.Research shows the key that the lymphocytic abundant activation of T is brain tumor immunity.The activation of T cell at least needs two signals: first signal by the antigen-specific receptor of T cell surface and the MHC antigenic peptide complexes of antigen presenting cell (APC) or tumor cell surface in conjunction with provide; Secondary signal is produced by the costimulatory molecules on APC (costimulatory molecules) and the corresponding receptors bind of T cell surface, and this signal can affect activation, propagation and the cytokine secretion of T cell.Costimulatory molecules can be divided into positivity and negativity, keep the inherent balance of positive and negative costimulatory signal for maintain body immune system function normal, avoid the unnecessary activation of T cell or suppress significant, and tumour cell often escapes from the immunosurveillance of body by low expression positivity costimulatory molecules or high expressed negativity costimulatory molecules.B7 family has comprised many important costimulatory molecules, as B7-1, B7-2, B7-H1 etc., can promote or suppressor T cell propagation and cell factor generation, and B cell activation, antibody are produced and also brings into play important regulative.The discoveries such as DM Kuang B7-H1 in liver cancer matrix is positive, and macrophage can suppress tumour-specific T cellular immunity, and gather density in liver cancer of this group of macrophage and the prognosis of hepatocarcinoma patient are negative correlation.For the B7-H4 recently finding, existing research has confirmed that brain Tumor Stem Cells (brain tumor stem cells) can pass through B7-H1/B7-H4 approach generation immunologic escape.

Along with going deep into that B7-H4 is studied, there is some scholars to start to be conceived to the relation of B7-H4 and tumor prognosis.Arigami etc. find the content of B7-H4 in tumor tissues and Patients with Gastric Cancer by stages, 5 years survival rate significant correlations, and can be used as the independent effect factor of cancer of the stomach prognosis, in addition, they have also studied the survival analysis of peripheral blood lymphocyte B7-H4 expression, and have obtained similar result.Chen etc. have carried out the SABC of squamous cell carcinoma of esophagus, have also confirmed that by analysis the tumour patient of B7-H4 high expressed presents worse prognosis.Kidney, melanoma field have also proved the importance of B7-H4 aspect tumor prognosis in succession.Wherein, the most noticeable is the research of Irish etc., they find that in oophoroma, the expression of B7-H4 is independent of CA125, if by serum B7-H4 separately for diagnosis, when specificity is 97%, sensitivity can reach 45%, if and B7-H4 is combined with CA125, sensitivity can be increased to 65% and be better than traditional CA125 diagnostic method.These all point out B7-H4 will can be used as a kind of novel tumor mark.

ELISA (enzyme linked immunosorbent assay (ELISA)) technology is that the antigen of solubility or antibody are adsorbed onto on the solid phase carriers such as polystyrene, carries out immunoreactive quantitative and qualitative analysis method.Its ultimate principle is first to prepare enzyme-labelled antigen or antibody, even if antigen or antibody are connected with certain enzyme, both retained its immunocompetence, the activity that retains again enzyme, then being examined sample (measuring antibody or antigen wherein) and enzyme-labelled antigen or antibody, by the antigen of different steps and surface of solid phase carriers or antibody, react, the antigen antibody complex forming on washing purifying solid phase carrier, the enzyme amount that makes to be combined on solid phase carrier becomes certain ratio with the amount of tested substance in sample, the substrate that finally adds enzyme reaction, the amount of coloured product that substrate is become by enzymatic is directly related with the amount of tested substance in sample, therefore can carry out qualitative or quantitative test according to the depth of color reaction.Therefore, in view of ELISA method have advantages of simple to operate, susceptibility is high, can be quantitative, applicant of the present invention intends by detect cerebrospinal fluid B7-H4 content with ELISA, set up and a kind ofly can carry out to patients with gliomas the method for early screening, diagnosis, antidiastole, this is all of great importance to the prevention of glioma, treatment.

Summary of the invention

The object of this invention is to provide new glioma tumor markers B7-H4, the present invention is the expression in gliomatosis human cerebrospinal fluid by research negativity costimulatory molecules B7-H4, has proposed B7-H4 and can be used as glioma tumor markers;

Another object of the present invention is to provide a kind of kit for glioma early diagnosis, in the present invention, B7-H4 glioma tumor markers for diagnosis of glioma, examination and evaluate its prognosis, can be solved to current glioma early diagnosis is difficult, antidiastole too relies on the problem of pathology and is applied to associated treatment scheme and prognosis evaluation.

The present invention studies show that, human brain tumour stem cell can be expressed B7-H4, the related experiment of mouse GL261 glioma cell line is disclosed to the positive glioma stem cells of the CD133 B7-H4 higher compared with the tumor cells expression of CD133 feminine gender, the obvious immunologic function of suppressor T cell in vitro, promote in vivo the growth of nude mice by subcutaneous transplantable tumor, research is demonstration also, and the microglia/macrophage of the CD11b positive is also expressed B7-H4.

Further, the present invention has carried out the clinical research of B7-H4, compares low level glioma express more B7-H4 protein by western blot discovery High Grade Gliomas, and qRT-PCR subsequently has also confirmed the above results from mRNA level.Further, in order to show more intuitively the expression of B7-H4 in tumor tissues, in the present invention, glioma sample to normal cerebral tissue and different stage has been implemented SABC and immunofluorescence dyeing, verified that B7-H4 is along with tumour rank increases increasing expression and the phenomenon expressed hardly in normal structure, the more important thing is, according to B7-H4 dyeing situation, 64 routine glioblastomas (WHO IV level) patient is being carried out after survival analysis, result shows, the patient of tumor tissues high expressed B7-H4 has relatively bad prognosis, prompting B7-H4 perhaps can be used in the prognosis of instructing glioma patient.

In view of microglia/macrophage is the main immunocyte in brain tissue, in the present invention, sorting purifying this class cell in brain tissue and peripheral blood, through flow cytometry, show, the B7-H4 positive microglial cells/macrophage in High Grade Gliomas tissue significantly raises.

The present invention is by detecting the content of B7-H4 in normal person, different stage patients with gliomas cerebrospinal fluid, utilizes statistical analysis to disclose the correlativity of B7-H4 and glioma, for the clinical osology of glioma, diagnoses and proposed B7-H4 and can be used as glioma tumor markers.

The invention discloses a kind of diagnosis of glioma kit and its preparation method and application, this kit can detect the B7-H4 in cerebrospinal fluid accurately and rapidly, and then can make auxiliary diagnosis timely to each phase glioma, has higher clinical value.

In the present invention, for the kit of glioma early diagnosis and antidiastole, comprise that mouse-anti people B7-H4 monoclonal antibody, horseradish peroxidase-labeled two is anti-, common reagent Tween-20, chromogenic substrate ABTS, ELISA Plate etc.This kit can quantitatively detect B7-H4 expressing quantity in cerebrospinal fluid, thus the ill risk of prediction glioma, examination people at highest risk, early diagnosis patients with gliomas, differentiates glioma and other intracranial space-occupying lesions.It is highly sensitive, can detect the B7-H4 of 30pg/ml, and specificity is high, does not have cross reaction with other cell factors of people, for the Accurate Diagnosis of glioma and control provide very strong technical foundation.

There is certain correlativity in the content of the B7-H4 based on described in cerebrospinal fluid and the tumour rank of patients with gliomas, therefore, kit of the present invention is also for assessment of the prognosis of glioma.

Accompanying drawing explanation

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

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

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

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

Fig. 5: the present invention is for diagnosing the ROC curve of glioma.

Embodiment

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

Embodiment 1,

One, Western blot detects normal cerebral tissue, different stage glioma B7-H4 expression:

Protein example preparation: add homogenate buffer after brain tissue shreds, machinery or ultrasound wave room temperature homogenate 0.5-1min.Then 4 ℃, the centrifugal 15min of 12,000g.Get supernatant as sample;

Electrophoresis: preparation SDS-PAGE running gel, in sample, add sample-loading buffer, be cooled to after room temperature, protein sample loading to gel is carried out to electrophoresis; Transferring film: adhesive tape is cut to suitable size after electrophoresis finishes, with transferring film damping fluid balance, 5min * 3 time.Cut out in advance the filter paper onesize with adhesive tape and NC film, immerse 10min in transferring film damping fluid; Membrane-transferring device is put well by the order of carbon anode plate, 24 metafiltration paper, NC film, gel, 24 metafiltration paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, NC film Accurate align, and each step is removed bubble, and upper ballast, blots liquid unnecessary on carbon plate; Switch on power, constant current 300mA, shifts 1.5h; After transfer finishes, deenergization takes out film;

Antibody incubation: cut film bar to be measured, wash film with 0.01M PBS, 5min * 3 time.Add confining liquid, steadily shake, room temperature 2h.Abandon confining liquid, with 0.01M PBS, wash film, 5min * 3 time.Add the anti-human B7-H4 primary antibodie of rabbit (1: 5000, Epitomics), 4 ℃ place 12h more than; Negative control, replaces primary antibodie with 1%BSA, and all the other steps are identical with experimental group; Abandon primary antibodie and 1%BSA, with 0.01M PBS, wash respectively film, 5min * 4 time.Add two of horseradish peroxidase to resist, steadily shake room temperature 2h.Abandon two and resist, with 0.01M PBS, wash film, 5min * 4 time;

Colour developing: add nitrite ion, prepare film in darkroom;

Shown in Fig. 1, known according to shade, High Grade Gliomas is expressed B7-H4 more than low level glioma, and normal cerebral tissue expresses hardly;

Two, immunohistochemical staining detects normal cerebral tissue, different stage glioma B7-H4 expression:

Dewaxing and aquation: brain tissue paraffin section is placed in to dimethylbenzene I 30min, 20min in dimethylbenzene II, 10min in dimethylbenzene III, in absolute ethyl alcohol, soak 5mins, in 95% ethanol, soak 5min, in 80% ethanol, soak 5mins, in 60% ethanol, soak 5min, single steaming in water soaked 5min;

Antigen retrieval: get a certain amount of CB damping fluid in a high-temperature resistant container, put in micro-wave oven power10 * 2min into, make it boiling, then section is carefully put into wherein, more slowly put into micro-wave oven and heat, power3 * 5min, last, be placed on the naturally cooling 30-40min of room temperature;

Dyeing: take out section, TBS rinses 4-5 time, and drying is cleaned.Add 30ul H2O2, in the wet box of room temperature, seal 30min, drying is cleaned; Drip mouse-anti people's B7-H4 primary antibodie (1: 50, AbD Serotec) 40ul, 4 ℃ are spent the night, TBST rinses 4-5 time, drips the anti-20ul of biotinylation two, incubated at room 1h, TBS rinses 4-5 time, drips enzyme mark Avidin 20ul, incubated at room 30min, TBS rinses 4-5 time, and dropping 30ul substrate is painted to be rinsed well rapidly afterwards, drips 1 haematine, 5-10min, dehydration, mounting, microscopy;

Shown in Fig. 2, brown color dyeing prompting B7-H4 positive cell, contrasts knownly, and High Grade Gliomas B7-H4 positive cell is maximum, and normal structure is only more than negative control;

Three, B7-H4 positive microglial cells ratio in Flow cytometry normal cerebral tissue, different stage glioma:

Separated microglia: get fresh brain tissue sample (in excision 20h), shred, centrifugal 1500rpm * 5min, abandon supernatant, add papain (1mg/ml) 4-5ml, 37 ℃ of digestion 30min, increase serum stops the rear centrifugal 1500rpm * 5min of digestion, abandon supernatant, resuspended with 1640 nutrient culture media of 10ml, cross 40 μ m filter screens in 15ml centrifuge tube, centrifugal 1500rpm * 5min, abandon supernatant, in centrifuge tube, add successively 70%Percoll solution 4ml, 30%Percoll solution 8ml, HBSS2ml, centrifugal 500g * 30min, from 70%, draw cell with the interface of 30%Percoll solution, the resuspended rear centrifugal 1500rpm * 5min of PBS, abandon supernatant,

Immunological magnetic bead sorting: with the resuspended upper step gained cell of MACS damping fluid of 80 μ l, the immunomagnetic beads 20 μ l that add coated anti-human CD11b antibody, 4 ℃ of 15min, MACS damping fluid washing 2 times, the centrifugal supernatant of abandoning, 500 μ l MACS damping fluid re-suspended cells, cross post, move post and go out magnetic field, with the MACS damping fluid of 1ml, wash post, collect washing lotion and be positive cell;

Flow cytometry: 100 μ l PBS re-suspended cells, add the anti-human CD11b antibody of 5 μ l APC combinations and the anti-human B7-H4 antibody of 5 μ 1PE combinations (1: 5, eBioscience), hatch 15min for 4 ℃, PBS washes 2 times, 300 μ l paraformaldehyde re-suspended cells, upper machine testing;

Shown in Fig. 3, the streaming figure upper right corner is B7-H4 positive microglial cells, and the B7-H4 positive cell ratio of High Grade Gliomas, low level glioma, normal cerebral tissue reduces successively.

The kit of embodiment 2, the early diagnosis of preparation glioma

Described kit comprises following component:

1, solid phase 96 hole ELISA Plate;

2, mouse-anti people B7-H4 purified monoclonal antibody (eBioscience);

3, mouse-anti people B7-H4 biotin labeling monoclonal antibody (eBioscience);

4, streptomysin avidin-peroxidase working fluid (Abcam);

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

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

7, cleansing solution: 0.05%Tween20-PBS;

8, dilution: 0.1%BSA-PBS;

9, stop buffer: 2M H2SO4.

The detection method of kit, comprising:

Standard items preparation: the B7-H4 standard items of different content are dissolved in to dilution, reach 0.1,0.5,2,10,25,50,100ng/ml isoconcentration;

Coated sealing: mouse-anti people B7-H4 purified monoclonal antibody is diluted to 2ug/ml, the coated 100ul's in the every hole of ELISA Plate, 4 ℃ are spent the night.Cleansing solution cleans 6 times, adds dilution 200ul/ hole, 37 ℃ of standing 1h;

Application of sample: cleansing solution cleaning of enzyme target 6 times, establish respectively blank well, gauge orifice, testing sample hole, blank well adds sample diluting liquid 100ul, and remaining hole adds respectively standard items or testing sample 50ul, and light rolling mixes, overlay film in ELISA Plate, 37 ℃ of reaction 2h;

Primary antibodie is hatched: discard liquid, dry, every hole adds 50ul biotin labeling B7-H4 antibody working fluid (1: 100), 37 degrees Celsius of standing 1h;

Enzyme labelled antibody is hatched: discards liquid, dries, wash 6 times, and each 1-2min, 350ul/ hole, dries, and every hole adds 50ul streptomysin avidin-peroxidase working fluid (1: 1000), 37 degrees Celsius of standing 0.5h;

Colour developing: cleansing solution cleaning of enzyme target 6 times, every hole adds 100ul ABTS substrate working fluid (1: 1000), 37 degrees Celsius of lucifuge colour developing 0.5h; Every hole adds 50ul stop buffer, cessation reaction;

Detection level: in the optical density value (OD value) in each hole of 450nm wavelength measurement, according to standard items OD value drawing standard curve, and then calculate the B7-H4 concentration of sample to be measured by microplate reader.

Embodiment 3, kit detect cerebrospinal fluid B7-H4 concentration for diagnosing sensitivity, the specificity of glioma

Make a collection of specimens: first obtain 2011-2012 and go to a doctor in 56 routine patients (the 5 routine brain traumas of Huashan hospital neurosurgery, 1 routine meningioma, 2 routine metastatic tumors, 16 routine low level gliomas, 32 routine High Grade Gliomas) after agreement, they have been carried out to waist and worn, everyone leaves and takes 5ml cerebrospinal fluid, is stored in-80 ℃ of refrigerators;

Detect B7-H4 concentration: the cerebrospinal fluid sample 30-60min that thaws in ice bath, centrifugal 1500rpm * 5min, get supernatant, according to the method in embodiment 2, adopt described kit, through standard items preparations, coated sealing, application of sample, primary antibodie hatch, the step such as enzyme labelled antibody is hatched, colour developing, microplate reader detection, measure the B7-H4 concentration of each sample;

Data statistics (as shown in Figure 4): use GraphPad5.02 software; Cerebrospinal fluid B7-H4 concentration determination result adopts mean value ± SEM to represent, can obtain non-glioma group 95.14 ± 16.30ng/ml, low level glioma group 123.2 ± 11.25ng/ml, High Grade Gliomas group 291.4 ± 29.51ng/ml, glioma group 235.3 ± 23.02ng/ml; Between group, adopt t check relatively, can obtain non-glioma group B7-H4 concentration is significantly lower than glioma groupand High Grade Gliomas group (P=0.0023) (P=0.0175), low level glioma group B7-H4 concentration is significantly lower than High Grade Gliomas group (P=0.0003), but not between glioma group and low level glioma group without significant difference (P=0.1669);

ROC tracing analysis: adopt SPSS Statistics20 software; Draw the ROC curve (as shown in Figure 5A) of diagnosis glioma, area under curve is 0.854, choose the maximum point of youden index (sensitivity+specificity-1) as the critical value of diagnosis glioma, result shows, when using cerebrospinal fluid B7-H4 concentration >=133.6170ng/ml as diagnosis glioma standard time, sensitivity is 70.8%, and specificity is 100%; Draw the ROC curve (as shown in Figure 5 B) that compares low level glioma and High Grade Gliomas, area under curve is 0.871, choose the point of youden index maximum as the critical value of diagnosis glioma, result shows, when usining cerebrospinal fluid B7-H4 concentration 135.1305ng/ml when differentiating the standard of low level glioma and High Grade Gliomas, sensitivity is 87.5%, and specificity is 75.0%.

The clinical practice of embodiment 4, kit

The preoperative waist that carries out of a certain doubtful patients with gliomas is worn, gather 4ml cerebrospinal fluid, method in reference example 2, adopt kit of the present invention, the steps such as the preparation of process standard items, coated sealing, application of sample, primary antibodie are hatched, enzyme labelled antibody is hatched, colour developing, microplate reader detection, measure its B7-H4 concentration;

The absorbance of reference standards, the B7-H4 concentration that draws this sample is 149.4877ng/ml; According to as described in Example 3 to 56 routine gliomas and the specified standard of non-gliomatosis human cerebrospinal fluid B7-H4 concentration determination, be that cerebrospinal fluid B7-H4 concentration >=133.6170ng/ml is probably glioma, and this glioma is probably high-level during concentration >=135.1305ng/ml, according to the above-mentioned B7-H4 concentration recording, diagnose this patient for High Grade Gliomas; Through postoperative pathological result, confirm, this patient is glioma WHO IV level.

Claims (7)

1. the purposes of cerebrospinal fluid B7-H4 albumen in preparing diagnosis of glioma preparation; Described B7-H4 albumen is as glioma tumor markers.

2. for a kit for glioma early diagnosis, it is characterized in that, comprise following component: solid phase 96 hole ELISA Plate; Mouse-anti people B7-H4 purified monoclonal antibody (eBioscience); Mouse-anti people B7-H4 biotin labeling monoclonal antibody (eBioscience); Streptomysin avidin-peroxidase working fluid (Abcam); B7-H4 protein standard substance (R & D Systems); ABTS substrate; 0.05%Tween20-PBS cleansing solution; 0.1%BSA-PBS dilution; Stop buffer.

3. a method that detects B7-H4 protein concentration in cerebrospinal fluid, is characterized in that, adopts the kit of claim 2 to detect B7-H4 protein concentration in sample to be tested cerebrospinal fluid, and it comprises step:

Protein standard substance preparation; Coated sealing mouse-anti people B7-H4; Standard items and sample to be tested application of sample; Primary antibodie is hatched; Enzyme labelled antibody is hatched; Substrate colour developing; Microplate reader detects; Drawing standard curve, calculating concentration of specimens.

4. by purposes claimed in claim 1, it is characterized in that, during cerebrospinal fluid B7-H4 protein concentration >=133.6170ng/ml, tentative diagnosis is glioma.

5. by purposes claimed in claim 1, it is characterized in that, during cerebrospinal fluid B7-H4 protein concentration >=135.1305ng/ml, tentative diagnosis is High Grade Gliomas.

6. the purposes of cerebrospinal fluid B7-H4 albumen in preparing different stage glioma antidiastole preparation; Described B7-H4 albumen is as glioma tumor markers.

7. the purposes of cerebrospinal fluid B7-H4 albumen in the preparation of preparation assessment glioma prognosis; Described B7-H4 albumen is as glioma tumor markers.