CN103439509B - Application of specific circulating non-small-cell lung cancer cell marker - Google Patents
Application of specific circulating non-small-cell lung cancer cell marker Download PDFInfo
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Abstract
The invention discloses an application of a specific circulating non-small-cell lung cancer cell marker, and relates to an application of a monoclonal antibody of SP70 in preparing a non-small-cell lung cancer peripheral blood circulating tumor cell detection regent. A flow cytometry kit for detecting non-small-cell lung cancer peripheral blood circulating tumor cells comprises a fluorescence labeled monoclonal antibody of SP70, a hemolytic agent and PBS (Phosphate Buffer Solution). According to the invention, lots of experiments indicate that the SP70 is capable of serving as a sensitive and specific detection index for the detection of the non-small-cell lung cancer peripheral blood circulating tumor cells, and can be used for detecting the non-small-cell lung cancer peripheral blood circulating tumor cells; as a result, the sensitivity and specificity of non-small-cell lung cancer diagnosis can be greatly improved; meanwhile the SP70 has excellent curative effect monitoring and prognosis effects, and also has high clinical application value.
Description
Technical field
The invention belongs to field of biological detection, relate to the application of specificity circulation non-small cell lung cancer cell mark.
Background technology
Lung cancer is the malignant tumour that in worldwide, M & M ranks first, and human health is caused to great threat, and has the trend increasing gradually.Lung cancer also occupies the first place of various malignant tumours at the M & M of China, approximately have every year 1200000 patients to die from lung cancer.In recent years, along with increasing the weight of of urban air pollution, particularly PM2.5 content continues to maintain high-order, the sustainable growth of population, the aggravation of aging population, adding degradation under smoking, occupational exposure and immunity of human body itself affects the increase of tumor invasion and dead hazards, and the M & M of lung cancer obviously rises.
According to different clinical manifestations and the sensitivity to chemicotherapy, lung cancer can be divided into two large classes, non-small cell lung cancer (non-small cell lung cancer, and small-cell carcinoma of the lung (small cell lung cancer NSCLC), SCLC), non-small cell lung cancer accounts for 80%～85% of lung cancer.When a lot of patients make a definite diagnosis, be late period, missed best operation period, cannot tolerate in addition after radiotherapy or chemotherapy and chemotherapy the reasons such as resistance, caused 5 years survival rates of lung cancer still to rest on 15% so low-down level nearly three in a decade or so.In the NSCLC of I phase underwent operative radical treatment, still have and exceed 30% patient and recurred in 5 years, and recurrence mostly is general and shifts, point out the early stage generation that just may have lung cancer micrometastasis (Micrometastasis) of existing clinical stages, and the methods such as conventional iconography, histology or cytology are difficult to find.It is one of committed step of tumour generation DISTANT METASTASES IN that tumour cell enters blood circulation.Therefore the research, patients with lung cancer specificity cyclic pulmonary cancer cell (specific circulating lung cancer cell, SCLCC) being detected has been subject to increasing attention.
1869, Ashworth found that a kind of haemocyte in blood is similar with the tumour cell of postmortem discovery, proposes first the concept of circulating tumor cell (circulating tumor cell, CTC).At present CTCs is defined as spontaneous or because operation of diagnosis and treatment departs from solid tumor primary tumor or metastasis and enter a class tumour cell of Peripheral Circulation, it seldom sees normal person.The CTCs not being eliminated through immune evasion not only can develop into metastasis under certain condition, and can form the micro-cancer embolus of circulation (circulating tumor microemboli, CTM) by migration, adhesion, mutual gathering.Current research shows that this small cancer embolus can also be attached to blood vessel endothelium, causes thrombosis, causes VTE (venous thromboembolism, VTE).Current metastases theory thinks, tumour cell needs first depart from and enter blood or Lymphatic Circulation from primary tumor, could form a long way off metastasis.Therefore, the hematogenous metastasis of CTCs and tumour has direct relation in theory, and further investigation CTCs contributes to the further understanding to mechanism of tumor metastasis, for the treatment of anti-tumor metastasis provides new foundation.
CTCs detects as finding the early stage new method shifting of tumour, without wound and as more reliable in clinical manifestation, Radiologic imaging and blood serum designated object than classic method.Diagnosis, the recurrence of monitoring postoperative patient tumour and the susceptibility of transfer, assessment antineoplastic that the detection of CTCs contributes to early stage metastatic tumour patient and patient's prognosis and select the therapeutic strategy of individuation, and originate from for the patient that cannot obtain tumor tissues late period molecules detection that the CTCs of solid tumor is its targeted therapy and opened one and fan gate.CTCs has been used to study kinds of tumors at present, as, the micrometastasis of breast cancer, prostate cancer, carcinoma of the colon and rectum, lung cancer etc. and guides clinical treatment.
But, for the determination and analysis of CTCs but and be not easy, clearly distinguish with other haemocyte because CTCs does not have significant specificity, and the tumour that different tissues is learned type and molecular phenotype is expressed respectively different marks; In addition, CTCs is quantity rareness in peripheral blood, and accurately separation is very difficult, has limited the application of CTCs.Along with people's going deep into mechanism of tumor metastasis research, especially follow the widespread use of modern detecting, CTCs is valued by the people gradually, current vast tumor research personnel special concern be susceptibility and the specificity that how to find the Specific marker of CTCs and improve detection means.Along with CTCs detection technique is updated, obtain some breakthrough progress, part detection method is all more satisfactory at aspects such as stability, susceptibility and specificitys, and clinical application research also makes great progress.
The method flow detecting for circulating tumor cell is at present all the concentration and analysis two-step approach based on to tumour cell.Generally speaking, CTC detects and generally includes beneficiation technologies and analytical technology.Beneficiation technologies is mainly got up these cell-specific ground enrichments by some physical and chemical principles, improves the susceptibility that CTCs detects.Analytical technology roughly can be divided into cell count and the technology based on nucleic acid, mainly relies on some tumour-specifics of existing on tumour cell or the morphological feature of organ specificity mark and tumour cell itself to find CTCs.In practical operation, often need to select the distinct methods of two parts in addition effectively combination, finally find out all higher methods of susceptibility and specificity.Because the step of manual method is various, cause poor repeatability, sensitivity is also very low, conventionally need to count 10
5～7can find 1 tumour cell.Testing staff's level of existing Technology Need and technology platform water mean height, therefore cost performance is low; Meanwhile, existing technology cannot realize the detection of specificity cyclic pulmonary cancer cell truly.So existing CTCs detection technique can not be used for the detection of specificity cyclic pulmonary cancer cell.
This laboratory is in earlier stage taking human lung adenocarcinoma cell line SPC-A1 as antigen, immunity BALB/c mouse, conventional Fusion of Cells, CELISA screening positive cell clone indirectly, obtains that a plant height is tired and the cell line NJ001 of the anti-human non-small cell lung carcinoma monoclonal antibody of stably excreting IgG1; Western blot shows that the antigen relative molecular mass (Mr) of monoclonal antibody NJ001 specific binding is 70000 left and right, and therefore this albumen is named as SP70; Immunofluorescence technique confirms that monoclonal antibody NJ001 can identify lung cancer cell line specifically, and its identification antigen all has expression in the endochylema of non-small cell lung cancer cell He on after birth, and in endochylema, expression is abundanter; Cell indirect ELISA testing result shows that monoclonal antibody NJ001 has specific reaction to adenocarcinoma of lung and squamous cell carcinoma strain, and with other kinds of tumor cell lines and normal cell is reactionless or hypoergia.ImmunohistochemistryResults Results shows that monoclonal antibody NJ001 and Non-Small Cell Lung Carcinoma have strong positive reaction, react and be weak positive or negative with the tumour of small-cell carcinoma of the lung, other organization types and normal lung tissue, therefore proved the specificity of SP70 in Diagnosis of Non-Small Cell Lung, and be greater than 85% Non-Small Cell Lung Carcinoma and have the expression of this specificity SP70 antigen.By double-antibodies sandwich ELISA, confirm that SP70 all has higher positive rate in NSCLC patient's serum and pleural effusion, can be used as the mark of NSCLC diagnosis and antidiastole, there is important clinical value.But whether SP70 expresses and there is not yet report in Peripheral Blood of Patients with Non-small Cell Lung circulating tumor cell.
At present, the clinical main tumor markers that is usually used in detecting serum of patients with non-small cell lung sample, has carcinomebryonic antigen (CEA), Cyfra21-1 segment (CYFRA21-1) and neuronspecific enolase (NSE).CEA is the acidoglycoprotein that a class has human embryos antigentic specificity determinant, is present on cell membrane, in Embryonic Stages and tumor tissues, expresses, and is non-specific tumor markers.CYFRA21-1 is a kind of Acid polypeptide, is mainly present in the kytoplasm of the epithelium genesis tumour cell such as lung cancer, oesophagus portion, and when normal, CK19 exists with oligomer form, and in serum, content is extremely low.NSE is a kind of acid protease, exists only in neuron and neuroendocrine cell.They are after the dissolving of tumour cell or necrosis, to come off to discharge into blood and cause extremely and increase; In the Malignant Pleural causing in lung cancer, also there is higher expression, all less than higher specificity.At present also not relevant report shows that they have specific expressed in the CTCs of lung cancer.Confirm that SP70 all has expression in the endochylema of non-small cell lung cancer cell He on after birth, in patient's serum and pleural effusion, all had higher positive rate.
Before finding SP70 at us, there is no to find to be applicable to the lung cancer tumor cell surface mark that lung cancer circulating tumor cell detects; Therefore, up to the present, not yet set up the detection technique of a lung cancer circulating tumor cell truly.Discovery and the development of SP70 albumen and specific recognition monoclonal antibody NJ001-1 thereof, for the foundation of the detection technique of specificity cyclic pulmonary cancer cell (specific circulating lung cancer cell, SCLCC) provides may.The present invention has proposed the concept that specificity cyclic pulmonary cancer cell detects first, and has set up corresponding detection new technology.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the application of SP70 as Peripheral Blood of Patients with Non-small Cell Lung circulating tumor cell mark is provided.
Another object of the present invention is to provide the application of the monoclonal antibody of SP70.
Another object of the present invention is to provide a kind of fluidic cell detection kit of Peripheral Blood of NSCLC Patients circulating tumor cell.
Object of the present invention can be achieved through the following technical solutions:
SP70 detects the application in reagent as Peripheral Blood of NSCLC Patients circulating tumor cell mark preparing Peripheral Blood of NSCLC Patients circulating tumor cell.
The monoclonal antibody of SP70 is in the application of preparing in Peripheral Blood of NSCLC Patients circulating tumor cell detection reagent.
The preferred preserving number of monoclonal antibody of described SP70 is the monoclonal antibody NJ001-1 of the anti-human non-small cell lung cancer of the hybridoma cell strain secretion of CCTCC NO:C201172.
Described monoclonal antibody NJ001-1 preferably detects the application in reagent at the fluidic cell of preparation detection Peripheral Blood of NSCLC Patients circulating tumor cell.
Detect a fluidic cell detection kit for Peripheral Blood of NSCLC Patients circulating tumor cell, the monoclonal antibody that comprises fluorescently-labeled SP70, hemolytic agent and PBS; Wherein, the monoclonal antibody of described SP70 is that preserving number is the monoclonal antibody NJ001-1 of the anti-human non-small cell lung cancer of the hybridoma cell strain secretion of CCTCC NO:C201172.
The preferred Mix – of the monoclonal antibody n-Stain of described fluorescently-labeled SP70
tMcF
tMthe monoclonal antibody NJ001-1 of 488A antibody labeling kit mark.
A kind of diagnostic method of non-small cell lung cancer, utilize described kit, detect the circulation non-small cell lung cancer cell that carries NJ001-1 specific recognition antigen SP70 in peripheral blood to be checked by Flow Cytometry, be specificity cyclic pulmonary cancer cell (specific circulating lung cancer cell, SCLCC), if can obtain positive fluorescence signal, and reach certain positive counting, illustrate in peripheral circulation blood to be checked and contain non-small cell lung cancer tumour cell, person to be checked suffers from non-small cell lung cancer; Detect the treatment curative effect that SCLCC is conducive to judge non-small cell lung cancer, and patient's prognosis.
The foundation of fluidic cell detection technique: the preparation of cell be in test tube, add 1ml SPC ?A1 cell or HFL cell (1 × 10
6/ ml), add 5 μ l monoclonal NJ001 fluorescence antibodies, room temperature lucifuge reaction 20min; Add 2mlPBS, 1500rpm/min, centrifugal 5min, repeats 2 times; Add 1mlPBS re-suspended cell; The preparation of peripheral blood sample is in test tube, to add 100 μ l peripheral bloods, adds 5 μ l monoclonal NJ001 fluorescence antibodies, room temperature lucifuge reaction 20min; Add 900 μ l hemolytic agents (BD FACS Lysing Solution, 10 ×), reaction 10min; Add 2mlPBS, 1500rpm/min, centrifugal 5min, repeats 2 times; Add 1mlPBS re-suspended cell.With the SPC after mark ?A1 cell, HFL cell, (WBC is 8 × 10 to healthy human peripheral blood
6/ ml) with flow cytometer detect 2 × 10
5individual peripheral blood cells, sets negative door and positive door according to NJ001 specific antigen expression.Respectively 43 routine Patients with Non-small-cell Lungs, the 15 routine benign disease patients of lung and 35 routine physical examination of healthy populations are detected to 2 × 10 with flow cytometer
5individual peripheral blood cells (PBMC), obtains the positive cell number of expressing NJ001 specific antigen, i.e. the number of CTCs.
Beneficial effect of the present invention:
The present invention finds by lot of experiments, SP70 can be used as the sensitivity being detected by Peripheral Blood of NSCLC Patients circulating tumor cell, special detection index, detect Peripheral Blood of NSCLC Patients circulating tumor cell simultaneously, can greatly improve susceptibility and the specificity of Diagnosis of Non-Small Cell Lung, perioperatively is had to good curative effect monitoring effect simultaneously, there is very high clinical value.
The positive number of non-small cell lung cancer CTCs with 43 routine Patients with Non-small-cell Lungs, the 15 routine benign disease patients of lung and 35 routine physical examination of healthy populations is made ROC curve, and corresponding area under curve is 0.831(standard error: 0.044; 95% credibility interval 0739～0.901), best critical value is 107, corresponding susceptibility is 65.12%, specificity is 92%.
Respectively to 35 routine Healthy Peoples, 15 routine lung benign lesions, 43 routine Peripheral Blood of Patients with Non-small Cell Lungs carry out the blind screening of non-small cell lung cancer CTCs and survey, the positive of the preliminary peripheral blood CTCs that finds Patients with Non-small-cell Lung detects number and is significantly higher than Healthy People and the benign disease patient of lung, P<0.05.
In 43 routine Patients with Non-small-cell Lungs, having 25 routine Patients with Non-small-cell Lungs all to carry out the blind screening of preoperative and postoperative CTCs surveys, after the positive of the preliminary peripheral blood CTCs that finds Patients with Non-small-cell Lung detects and is significantly higher than operation before number operation, P<0.05.
It is 65.12% to 43 routine NSCLC patients' CTCs positive rate recall rate that fluidic cell of the present invention detects CTCs method, higher than CEA, the CYFRA21 of ECLIA method ?1 and the 34.88%(χ of NSE
2=5.76, P<0.05), 13.95%(χ
2=16.96, P<0.05) and 23.26%(χ
2=13.14, P<0.05), and higher than CEA, CYFRA21 ?1 and the positive rate 60.47% of tri-joint-detection of NSE, difference not statistically significant, P>0.05.By the method with for joint-detection, result shows, CTCs, CEA, CYFRA21 ?1 and the positive rate of tetra-joint-detection of NSE be 81.40%, higher than CEA, CYFRA21 ?1 and positive rate (60.47%, the χ of tri-joint-detection of NSE
2=7.11, P<0.05).
In sum, the monoclonal antibody of SP70 can be used for preparing Peripheral Blood of NSCLC Patients circulating tumor cell and detects reagent, for the diagnosis of non-small cell lung cancer.
The preservation information of biological material specimens
Hybridoma cell strain NM001-1 is preserved in Chinese Typical Representative culture collection center on August 31st, 2011, and preservation address is Wuhan, China, Wuhan University, and preserving number is CCTCC NO:C201172.
Brief description of the drawings
Fig. 1 fluorescence labeling NJ001 fluidic cell detects figure.A～C figure is unmarked fluorescence antibody fluidic cell testing result, A: Healthy People PBMC is unmarked, and B:HFL is unmarked, and C:SPC-A1 is unmarked; D～F figure is fluidic cell testing result after mark fluorescent antibody, D: after Healthy People PBMC mark, after E:HFL mark, after F:SPC-A1 mark.
The each composition flow cytometric analysis of Fig. 2 fluorescence labeling NJ001 periphery PBMC figure.Healthy People periphery PBMC is carried out to flow cytometer showed, analyze the fluorescence labeling situation of lymphocyte, monocyte and neutrophil leucocyte NJ001.Three the little figure in left side are that each component is established door, and middle three little figure are that each component is in contrast unmarked, and the little figure in three, right side is result after each component mark.
Fig. 3 SPC ?A1 cell and HFL cell press 1:10,1:100,1:1000, the ratio of 1:10000 adds fluorescence antibody mark, flow cytometer testing result after mixing.
A figure is HFL contrast, and B figure is SPC-A1 contrast, and C figure～F figure is followed successively by SPC-A1:HLF1:10,1:100,1:1000, the mixing of 1:10000 ratio.
Fig. 4 SPC ?A1 cell and 0.1ml healthy human peripheral blood press 1:10,1:100,1:1000, the ratio of 1:10000 adds fluorescence antibody mark, flow cytometer testing result after mixing.
A figure is PBMC contrast, and B figure is SPC-A1 contrast, and C figure～F figure is followed successively by SPC-A1:PBMC1:10,1:100,1:1000 and 1:10000 ratio to be mixed.
Fig. 5 SPC ?A1 cell and healthy human peripheral blood (WBC:6 × 10
9/ L) carry out sorting testing result in flow cytometer after the ratio mixing of 1:100
A figure is the SPC-A1 cell HE dyeing photo before sorting, and B figure is the SPC-A1 cell HE dyeing photo after sorting, and C figure is the common light microscopic photo of the SPC-A1 cell after sorting, and D figure is the SPC-A1 cell fluorescence photo after sorting.Fig. 6 SPC ?A1 cell and healthy human peripheral blood (WBC:5.5 × 10
9/ L) carry out sorting testing result in flow cytometer after the ratio mixing of 1:10
A figure is the SPC-A1 cell HE dyeing photo before sorting, and B figure is the SPC-A1 cell HE dyeing photo after sorting, and C figure is photo after the dyeing of SPC-A1 cell fluorescence, and D figure is the SPC-A1 cell fluorescence photo after sorting.
The ROC curve of Fig. 7 Patients with Non-small-cell Lung periphery CTCs
Area under curve is 0.831(standard error: 0.044; 95% credibility interval 0739～0.901)
NJ001 specific antigen CTC flow cytometer detection result comparison in Fig. 8 Healthy People, lung's benign disease and non-small cell lung cancer, wherein * represents P<0.05.
NJ001 specific antigen CTCs flow cytometer detection result comparison (n=25) before and after Fig. 9 Patients with Non-small-cell Lung corrective surgery.Wherein * represents P<0.05
Embodiment
Embodiment 1
Fluidic cell detects reagent: the monoclonal antibody, hemolytic agent (BD FACS Lysing Solution) and the PBS that comprise fluorescently-labeled SP70, wherein, the monoclonal antibody of fluorescently-labeled SP70 is Mix – n-Stain
tMcF
tMthe monoclonal antibody NJ001-1 of 488A antibody labeling kit mark.
The mark of fluorescence antibody: get 20 μ l monoclonal antibody NJ001(5 μ g/ μ and l) add in reaction column, 14000rpm/min, 4 DEG C, centrifugal 3min, discards from going out liquid; In reaction column, add 100 μ l PBS, 14000rpm/min, 4 DEG C, centrifugal 3min, discards from going out liquid; In reaction column, add the resuspended antibody of 100 μ l PBS, 10 × reactant liquor of getting 90 μ l antibody and 10 μ l mixes; Aforesaid liquid is all moved in fluorescent dye pipe and mixed, room temperature lucifuge reaction 30min; Again reacted whole liquid is moved in storage liquid pipe and is mixed, packing ， ?20 DEG C of preservations.
The foundation of fluidic cell detection technique: the foundation of fluidic cell detection technique: the preparation of cell be in test tube, add 1ml SPC ?A1 cell or HFL cell (1 × 10
6/ ml), add 5 μ l monoclonal NJ001 fluorescence antibodies, room temperature lucifuge reaction 20min; Add 2mlPBS, 1500rpm/min, centrifugal 5min, repeats 2 times; Add 1mlPBS re-suspended cell; The preparation of peripheral blood sample is in test tube, to add 100 μ l peripheral bloods, adds 5 μ l monoclonal NJ001 fluorescence antibodies, room temperature lucifuge reaction 20min; Add 900 μ l hemolytic agents (BD FACS Lysing Solution, 10 ×), reaction 10min; Add 2mlPBS, 1500rpm/min, centrifugal 5min, repeats 2 times; Add 1mlPBS re-suspended cell.With the SPC after mark ?A1 cell, HFL cell, (WBC is 8 × 10 to healthy human peripheral blood
6/ ml) with flow cytometer detect 2 × 10
4individual peripheral blood cells, sets negative door and positive door according to NJ001 specific antigen expression.
Adopt fluidic cell of the present invention detect reagent to respectively to healthy human peripheral blood, HFL cell (human embryonic lung cell) and SPC ?A1 cell (human lung adenocarcinoma cell) carry out the detection of SP70 antigen, SPC ?A1 cell detection to fluorescence intensity be significantly higher than HFL cell and healthy human peripheral blood cell (Fig. 1).Instruction book clonal antibody NJ001-1 to SPC ?the detection of A1 cell there is very high specificity.
Adopt fluidic cell of the present invention to detect reagent to respectively healthy human peripheral blood leucocyte composition being carried out to the detection of SP70 antigen, find that neutrophil leucocyte, monocyte and lymphocytic SP70 antigen are without expressing or low expression (Fig. 2).
Embodiment 2
Adopt fluidic cell of the present invention to detect reagent, get respectively SPC ?A1 cell and HFL cell by 1:10,1:100,1:1000, after the ratio of 1:10000 is mixed, adds the monoclonal antibody (with embodiment 1) of fluorescently-labeled SP70, after washing, with flow cytometer detection, the results are shown in Figure 3.The fluorescence antibody of the monoclonal antibody NJ001 of result show needle to SP70 albumen to SPC ?A1 cell have specific reaction, and can detect corresponding positive rate according to different blending ratios.
With fluidic cell detection reagent of the present invention, get respectively SPC ?A1 cell and 0.1ml healthy human peripheral blood by 1:10,1:100,1:1000, after the ratio of 1:10000 is mixed, add the monoclonal antibody (with embodiment 1) of fluorescently-labeled SP70, after haemolysis washing, with flow cytometer detection, the results are shown in Figure 4.Result shows: for the fluorescence antibody of the monoclonal antibody NJ001 of SP70 albumen to SPC ?A1 cell have specific reaction and can detect corresponding positive rate according to different blending ratios.
Embodiment 3
Adopt fluidic cell of the present invention to detect reagent, get respectively SPC ?A1 cell and healthy human peripheral blood (WBC:6 × 10
9/ L) mix in the ratio of 1:100 after, add fluorescence antibody mark (with embodiment 1), after haemolysis washing, carry out sorting detection with flow cytometer, the results are shown in Figure 5.Sorting 1.2 × 10
6after individual cell, obtain 2 × 10
4individual cell, obtains cell/sorting cells and is about 1/60.For the fluorescence antibody of the monoclonal antibody NJ001 of SP70 albumen to SPC ?A1 cell have specific reaction, the cell sub-electing more than 90% for SPC ?A1 cell.
Adopt fluidic cell of the present invention to detect reagent, get respectively SPC ?A1 cell and healthy human peripheral blood (WBC:5.5 × 10
9/ L) mix in the ratio of 1:10 after, add fluorescence antibody mark (with embodiment 1), after haemolysis washing, carry out sorting detection with flow cytometer, the results are shown in Figure 6.Sorting 2.8 × 10
6after individual cell, obtain 3 × 10
5cell, obtains cell/sorting cells and is about 1/9.3.For the fluorescence antibody of the monoclonal antibody NJ001 of SP70 albumen to SPC ?A1 cell have specific reaction, the cell sub-electing more than 90% for SPC ?A1 cell.
Embodiment 4
The foundation of fluidic cell detection technique: the preparation of cell be in test tube, add 1ml SPC ?A1 cell or HFL cell (1 × 10
6/ ml), add 5 μ l monoclonal NJ001 fluorescence antibodies, room temperature lucifuge reaction 20min; Add 2mlPBS, 1500rpm/min, centrifugal 5min, repeats 2 times; Add 1mlPBS re-suspended cell; The preparation of peripheral blood sample is in test tube, to add 100 μ l peripheral bloods, adds 5 μ l monoclonal NJ001 fluorescence antibodies, room temperature lucifuge reaction 20min; Add 900 μ l hemolytic agents (BD FACS Lysing Solution, 10 ×), reaction 10min; Add 2mlPBS, 1500rpm/min, centrifugal 5min, repeats 2 times; Add 1mlPBS re-suspended cell.Respectively 43 routine Patients with Non-small-cell Lungs, the 15 routine benign disease patients of lung and 35 routine physical examination of healthy populations are detected to 2 × 10 with flow cytometer
5individual peripheral blood cells (PBMC), obtains the positive cell number of expressing NJ001 specific antigen, i.e. the number of CTCs.
The positive number of CTCs with 43 routine Patients with Non-small-cell Lungs, the 15 routine benign disease patients of lung and 35 routine physical examination of healthy populations is made ROC curve, sees Fig. 7, and corresponding area under curve is 0.831(standard error: 0.044; 95% credibility interval 0739～0.901), best critical value is 107, corresponding susceptibility is 65.12%, specificity is 92%.
Embodiment 5
Respectively to 35 routine Healthy Peoples, 15 routine lung benign lesions, 43 routine Peripheral Blood of Patients with Non-small Cell Lungs carry out the blind screening of CTCs and survey, fluidic cell detection method is with embodiment 4, the results are shown in Figure 8, the positive of the preliminary peripheral blood CTCs that finds Patients with Non-small-cell Lung detects number and is significantly higher than Healthy People and the benign disease patient of lung, P<0.05.
Embodiment 6
In 43 routine Patients with Non-small-cell Lungs, having 25 routine Patients with Non-small-cell Lungs all to carry out the blind screening of preoperative and postoperative CTCs surveys, fluidic cell detection method is with embodiment 4, the results are shown in Figure 9, after the positive of the preliminary peripheral blood CTCs that finds Patients with Non-small-cell Lung detects and is significantly higher than operation before number operation, P<0.05.
Embodiment 7
Adopt Electrochemiluminescince (ECLIA method) detect CEA, CYFRA21 in serum ?1, NSE: by Roche MODULAR E170 electrochemiluminescence immunity analysis instrument and matched reagent thereof to 43 routine NSCLC patients carry out change of serum C EA, CYFRA21 ?1, the synchronous detection of NSE.
Flow cytometry is 65.12% to 43 routine NSCLC patients' CTCs positive rate recall rate, higher than CEA, the CYFRA21 of ECLIA method ?1 and the 34.88%(χ of NSE
2=5.76, P<0.05), 13.95%(χ
2=16.96, P<0.05) and 23.26%(χ
2=13.14, P<0.05), and higher than CEA, CYFRA21 ?1 and the positive rate 60.47% of tri-joint-detection of NSE, difference not statistically significant, P>0.05.By the method with for joint-detection, result shows, CTCs, CEA, CYFRA21 ?1 and the positive rate of tetra-joint-detection of NSE be 81.40%, higher than CEA, CYFRA21 ?1 and positive rate (60.47%, the χ of tri-joint-detection of NSE
2=7.11, P<0.05).
Individual event and the joint detection results positive of CTCs and three kinds of Tumor invasions in 43 routine non-small cell lung cancer groups
［ routine (%) ］
Claims (1)
1.SP70 detects the application in reagent as Peripheral Blood of NSCLC Patients circulating tumor cell mark preparing Peripheral Blood of NSCLC Patients circulating tumor cell.
The monoclonal antibody of 2.SP70 is in the application of preparing in Peripheral Blood of NSCLC Patients circulating tumor cell detection reagent, and the monoclonal antibody of described SP70 is that preserving number is the monoclonal antibody NJ001-1 of the anti-human non-small cell lung cancer of the hybridoma cell strain secretion of CCTCC NO:C201172.
3. application according to claim 2, is characterized in that the fluidic cell that described monoclonal antibody NJ001-1 detects Peripheral Blood of NSCLC Patients circulating tumor cell in preparation detects the application in reagent.
4. detect a fluidic cell detection kit for Peripheral Blood of NSCLC Patients circulating tumor cell, it is characterized in that the monoclonal antibody, hemolytic agent and the PBS that comprise fluorescently-labeled SP70; Wherein, the monoclonal antibody of described SP70 is that preserving number is the monoclonal antibody NJ001-1 of the anti-human non-small cell lung cancer of the hybridoma cell strain secretion of CCTCC NO:C201172.
5. kit according to claim 4, is characterized in that the monoclonal antibody of described fluorescently-labeled SP70 is Mix – n-Stain
tMcF
tMthe monoclonal antibody NJ001-1 of 488A antibody labeling kit mark.
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