CN109116023A - A kind of lung cancer marker anti-MM P12 autoantibody and its application - Google Patents
A kind of lung cancer marker anti-MM P12 autoantibody and its application Download PDFInfo
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Abstract
The invention belongs to technical field of biomedical detection, and in particular to a kind of lung cancer marker anti-MM P12 autoantibody and its application, the invention discloses a kind of lung cancer marker, the lung cancer marker is anti-MM P12 autoantibody.The present invention also provides above-mentioned lung cancer marker in preparation for the application in lung cancer detection kit.The present invention is intended to provide one kind is easy to operate, at low cost, accuracy is high, the kit for being applied to clinical lung cancer marker and the marker and evaluating in preparation for lung cancer of hurtless measure.The research of the invention finds that carrying out the detection of serum anti-MM P12 autoantibody using ELISA method, it can be accurately by patients with lung cancer and normal person, and benign tuberculosis group (COPD and chronic bronchitis) patient distinguishes and, and can be used for the early detection of lung cancer.The marker and the marker that there is provided this convenient, fast, effective detection patients with lung cancer in this context are used for the kit of lung cancer detection in preparation, can be used for clinical lung cancer early diagnosis.
Description
Technical field
The invention belongs to technical field of biomedical detection, and in particular to a kind of lung cancer marker anti-MM P12 autoantibody
And its application.
Background technique
Lung cancer is the malignant tumour of China and global incidence and the death rate first.Between past 40 years, 5 years of lung cancer
Survival rate only rises to 16% from 12%, main the reason is that when diagnosis has belonged to advanced stage, on the contrary, the lung cancer of early diagnosis carries out hand
Survival rates can be improved to 80%.As it can be seen that early discovery, the treatment and prognosis that early diagnose to lung cancer are anticipated with important clinic
Justice.The detection means of current extensive utilization includes noninvasive test (such as x-ray, CT, mammography in nonpalpable breast) and invasive inspection (fiber branch gas
Guan Jing, bronchography, B ultrasound or CT position lower aspiration biopsy etc.), but lack compliance and the universal possibility used.It looks for new
Lung cancer molecular marker, especially serum molecules marker, patients with lung cancer timely and effectively can early be looked into, early diagnosis, it is early control,
It is the key scientific problems for improving patients with lung cancer survival rate, reducing the death rate.Although having some tumor markers at present, such as
CA125 (cancer antigen 125), CA19-9 (CA 19-9), CEA (carcinomebryonic antigen) etc. can be used for the detection of lung cancer, but sensibility
It is not high with specificity, so so far, still not preferably for the lung cancer early screening and diagnostic markers of clinical use
Object.
Non-small cell lung cancer (non-small cell lung cancer, NSCLC) be lung cancer two big organization types it
One, account for about the 80% of lung cancer sum.It is 70-by I phase patient, 5 years survival rates of operative treatment by taking non-small cell lung cancer as an example
90%, and IV phase patient is only capable of alleviating clinical symptoms by palliative treatment mostly, survival rate is extremely low within 5 years.Therefore, early to find, is early
Diagnosis is the key point for reducing lung cancer mortality, improving survival of patients situation, so, find effective early diagnosis technology and
Method is most important.Currently, clinically mostly use imageological examination detection and diagnosis lung cancer, such as C-XF, Multi-section CT, low
Dose helical CT(low-dose computed tomography, LDCT) etc., but these Image Examinations usually have
Higher false positive rate, needs follow-up studies, for suspicious patient, often results in excessive radioactive exposure, while in various degree
Ground increases the psychology and financial burden of patient.2011, a randomized control study knot of American National screening lung cancer test
Fruit shows, compared with x-ray rabat, lung cancer case fatality rate decline 20% can be made by carrying out screening to High Risk of Lung Cancer crowd using LDCT, but
It still has deficiency, i.e. LDCT can have found tubercle in 25% people at highest risk, and pass through further inspection and tracking, finally quilt
It is diagnosed as only the 4% of lung cancer, and only 30% patients with lung cancer meets people at highest risk's screening standard of LDCT, illustrates major part
Patients with lung cancer can not be found by LDCT screening.In recent years, tumor serology marker is easy to operate due to traumatic small,
The advantages that screening range is wide, and detection efficiency is high is increasingly subject to the extensive concern of tumor area researcher.
Immune system plays indispensable role in the occurrence and development of tumour.After malignant tumour occurs, tumour is thin
Extracellular antigen is capable of the cell and immunity system of human activin, generates the autoantibody of anti-tumor cell antigen.These activation
The antigen of human immune system is known as tumor associated antigen (tumor associated antigen, TAA), their essence is
It discharges, fall off or exocrine differential protein product after meronecrosis or apoptosis during tumorigenesis.Face at present
In bed work, the more common tumor associated antigen in pulmonary cancer diagnosis has a CYFRA21-1, CEA, NSE, SCC etc., but tumour phase
It closes antigen to be easily degraded or remove in serum, short there are the time and lower to lung cancer early diagnosis sensibility, specificity is also not
Foot, thus it is limited for discovery and diagnosis early stage of lung cancer value.And the studies have shown that of the past is in tumorigenic early stage, based on exempting from
The signal amplifying function of epidemic disease system, even if only low-level tumour antigen is released into serum, the immune system of body can
It identifies these tumour specific antigens and generates corresponding autoantibody, these antibody are known as anti-TAAs autoantibody.Because having
Features, the anti-TAAs autoantibodies such as early stage can be detected, sample is stable, higher specificity, detection method are simple are considered having
Hoping becomes blood serum tumor markers of new generation.Studies have found that 5 years before autoradiographic technique detects non-small cell lung cancer
Antibodies to tumor-associated antigen (taa) can be detected in patient's body.Existing many cases report domestic at present finds other entity tumors, example
As breast cancer, liver cancer, oophoroma are detected the presence of anti-TAAs autoantibody.These research achievements prompt tumour correlation anti-
The detection of former autoantibody is expected to the important Serology biological marker as detection of early lung cancer.It is examined in view of current autoantibody
Survey still has deficiency in the clinical application of pulmonary cancer diagnosis, constantly finds that TAAs related to new lung cancer is identified is still one important
Work.
In conclusion improve survival rate to finally realize the death rate for reducing lung cancer, there is an urgent need in the art to screening and
It identifies more sensitive, specific serum autoantibody markers, and develops inspection easy to operate, at low cost, applied widely
Survey the kit of lung cancer autoantibody.
Summary of the invention
It is an object of the invention to overcome the problems, such as that the existing tumor markers of lung cancer are undesirable, it is intended to improve lung cancer early stage and examine
Disconnected sensitivity, specificity and accuracy.Good using the strong diagnosis marker preparation stability of species specificity height, susceptibility,
Kit for detection of early lung cancer easy to detect.The present invention specifically provides a kind of lung cancer early sign object, and in particular to
A kind of anti-MM P12 (Matrix metallopeptidase 12) autoantibody that can distinguish lung cancer and normal person.
To achieve the above object, the invention adopts the following technical scheme:
In a first aspect, the lung cancer marker is anti-MM P12 autoantibody the present invention provides a kind of lung cancer marker.
Second aspect, the present invention provides the anti-MM P12 autoantibodies described in first aspect to be used for Sera of Lung Cancer in preparation
Learn the application in the kit of detection.
Preferably, the kit is the detection architecture based on enzyme-linked immunosorbent assay (ELISA).
Preferably, the kit contains the MMP12 recombination purifying protein of detection anti-MM P12 autoantibody.
Preferably, the kit also contains ELISA coating buffer (20 ×), Block buffer (BSA), HRP-
Rec-A secondary antibody, ELISA universal antibody dilution and ELISA-ABTS colour reagent, PBST(polysorbas20-phosphate buffer),
Terminate liquid.
The beneficial effects of the present invention are:
The present invention is intended to provide one kind is easy to operate, at low cost, accuracy is high, the lung cancer marker for being applied to clinic of hurtless measure
And the marker is used for the kit of lung cancer detection in preparation.The research of the invention finds that it is anti-to carry out serum using ELISA method
The detection of MMP12 autoantibody, can be accurately by patients with lung cancer and normal person and benign tuberculosis group (COPD and chronic
Bronchitis) patient distinguishes and, and can be used for the early detection of lung cancer.It is convenient, fast, effective that this is provided in this context
Detection patients with lung cancer marker and the marker kit of lung cancer detection is used in preparation, it is early to can be used for clinical lung cancer
Phase diagnosis.
Detailed description of the invention
Fig. 1 is the Technology Roadmap of screening, identification and evaluation lung cancer anti-MM P12 autoantibody;
Fig. 2 is distribution of the anti-MM P12 autoantibody in lung cancer group and Normal group in test group;
Fig. 3 is anti-MM P12 autoantibody in test group to the ROC curve of lung cancer detection;
Fig. 4 is distribution (* of the anti-MM P12 autoantibody in lung cancer group, benign tuberculosis group and Normal group in validation groupP<
0.01);
Fig. 5 be validation group anti-MM P12 autoantibody lung cancer group respectively with Normal group, benign tuberculosis group and non-lung cancer group
ROC curve (the LC: lung cancer group compared;B: benign tuberculosis group: N: Normal group).
Specific embodiment
As shown in Figure 1, the present invention screens lung cancer associated tumor antigen using Oncomine database, and apply ELISA
Method detects anti-MM P12 antibody level in Serum of Patients with Lung Cancer, finally using ELISA method verifying anti-MM P12 to lung cancer
Detection value.
1) gene of Oncomine database mining unconventionality expression in cancerous lung tissue is utilized, and is filtered out in non-small cell
MRNA expression is apparently higher than the gene of normal lung tissue in cancerous lung tissue, and it may be candidate biological in lung cancer detection for prompting them
Marker.
2) it can be used as pulmonary cancer diagnosis marker for further verifying anti-MM P12 autoantibody, using the detection side ELISA
Method carries out the detection of anti-MM P12 autoantibody, step to 184 Serum of Patients with Lung Cancer and 184 normal human serums
Suddenly include: that the MMP12 albumen of purifying is diluted to 0.5ug/ml with antigenic dilution, be coated with 96 orifice plates, serum sample 1:100 is dilute
Release, after antigenic action, reacted with the goat anti-human igg antibody of horseradish peroxidase-labeled, ultimately join ABTS substrate solution into
Row colour developing.OD value is read, under microplate reader (405nm absorbance) to react the level of anti-MM P12 autoantibody in serum.Point
Not Li Yong two independent samples t tests and Mann-Whiteny U examine to the mean value of lung cancer group and Normal group antibody level and
Median is compared, and the distribution of anti-MM P12 autoantibody and is compared as shown in Fig. 2, its own antibody level in two groups
Difference between lung cancer group and Normal group it is statistically significant (P< 0.05), and for lung cancer group be higher than Normal group (P
< 0.05).The present invention is by carrying out ROC curve analysis to lung cancer group and Normal group, such as Fig. 3 the results show that lung cancer is to normal
The area under the curve of people is 0.70, and sensitivity and specificity are respectively 20.70% and 90.80%.
It 3) is to further appreciate that application value of the anti-MM P12 autoantibody in lung cancer detection, the present invention determine 446
The OD of anti-MM P12 autoantibody in example Serum of Patients with Lung Cancer, 446 normal control serum and 119 benign consumptive's serum
Value.As a result as shown in figure 4, comparing lung cancer group, benign tuberculosis group using one-way analysis of variance and Kruskal-Wallis inspection
There are indifference, the mean and median result of autoantibody with the mean value and median of Normal group autoantibody
Difference is statistically significant (P< 0.01).When distinguishing patients with lung cancer and normal control, the AUC of anti-MM P12 is 0.64
(95%CI:0.608-0.680,P< 0.001), i.e., this index can distinguish patients with lung cancer and normal control, and anti-MMP12 is certainly
Body antibody can also distinguish lung cancer and benign tuberculosis, AUC 0.68(95%CI:0.627-0.732,P< 0.001).In addition, with non-
Lung cancer group (benign tuberculosis group+Normal group) is 0.601(95%CI:0.567- compared to the AUC of anti-MM P12 autoantibody
0.636,P< 0.001), the results show that expression of the anti-MM P12 autoantibody in Serum of Patients with Lung Cancer is higher than benign lung
Patient and normal human serum have statistical significance (P < 0.001, as shown in Figure 5).Therefore, anti-MM P12 autoantibody energy
Patients with lung cancer and non-lung cancer (benign tuberculosis group+Normal group) crowd are distinguished to a certain extent.
Below by specific embodiment, the invention will be further described.
1. serum specimen is collected
184 Serum of Patients with Lung Cancer and 184 matched normal human serums of age-sex are used in the present invention, carry out anti-MM P12
Preliminary assessment of the autoantibody in pulmonary cancer diagnosis.Other 446 Serum of Patients with Lung Cancer, 119 lung's benign disease patient's blood
The matched normal human serum of clear and 446 age-sexes carries out the evaluation that value is further detected in the early stage of lung cancer.It is all to receive
Enter patients with lung cancer of the invention to make a definite diagnosis by histopathology, and without chemicotherapy.Normal control serum and clinical data
2013 to 2016 are collected in the medical patient of the first affiliated hospital of Zhengzhou University and physical examination of healthy population, all non-cancer subjects
It is verified to be not suffering from tumor-related illness and autoimmune disease.
All serum specimens in the present invention use inertia separation gel to promote solidifying pipe acquisition 3 ~ 4 ml of research object whole blood, put
After setting 4 DEG C of refrigerator 2h, 3500 rpm are centrifuged 5min, are dispensed into Eppendorf and mark specimen types and number for serum,
It is saved in -80 DEG C of refrigerators, avoids multigelation.
2. the screening of candidate lung cancer TAAs
This project early period is excavated and is screened to lung cancer gene-correlation information in Oncomine, main to utilize specific several sieves
Condition is selected to excavate compared with normal lung tissue, the obvious highly expressed gene in Non-Small Cell Lung Carcinoma.Specific screening item
Part is as follows: (1) Analysis Type:Cancer vs.Normal Analysis;(2) Cancer Type:Lung
Cancer--Non-Small Cell Lung Carcinoma;(3) Date Type:mRNA;(4) sample size is all larger than 100.Root
Three data sets, respectively Hou Lung, Landi Lung and Okayama Lung are picked out according to conditions above.As shown in table 1,
Three data collect into specimen amount be respectively 156,107 and 246, the chip type used is Human Genome U133
2.0 Array of Array or Human Genome U133 Array Plus.It is high in Non-Small Cell Lung Carcinoma to search out
Expression and difference have the gene of statistical significance, we carry out comprehensive analysis to these three data sets, and filter out in P value
Position rank is located at preceding 40 genes.MMP12 is concentrated in three dataPValue is respectively less than inspection level (0.05/ core after correcting
Piece detects number of genes) and differential expression multiple be all larger than 3, therefore anti-MMP12 autoantibody is further analyzed.
The essential information of three lung cancer associated data sets in 1 Oncomine of table
3. the serum expression of ELISA method detection anti-MM P12 autoantibody
Reagent needed for 3.1
(1) 1 × coating buffer: taking 2.5ml ELISA coating buffer (20 ×) into 50ml centrifuge tube, and deionization is added
Water is settled to 50ml, stirs, and is placed in 4 DEG C of preservations.
(2) 10 × PBST buffers: 81.8g sodium chloride, 3.1g NaH are weighed2PO4-2H2O、28.8g Na2HPO4-
12H2O, 0.1g merthiolate, 5ml Tween20 add deionized water to be settled to 1L, are transferred in liquid storage bottle in 1L graduated cylinder, mix
Room temperature preservation after closing uniformly.
(3) 1 × PBST buffers: taking 10 × PBST buffer 100ml in 1L graduated cylinder, deionized water added to be settled to 1L,
It is transferred in liquid storage bottle bottle, concussion mixes, and marks title and date, room temperature preservation.
3.2 anti-MM P12 autoantibody detection methods
The early-stage preparations of ELISA experiment: with 1 × be coated with buffer by the albumen of the commercialization of purchase in Eppendorf pipe into
Row dissolution, dilutes final concentration of 0.5 μ g/ μ l, posts protein tag and date, -20 DEG C of preservations respectively;It is taken from -80 DEG C of refrigerators
The case group and control group serum that early period collects out, difference sequencing numbers, with ELISA universal antibody dilution in 96 hole deep-well plates
In serum is diluted by 1:100, and marked in deep-well plates, 4 DEG C of preservations;It, will with 1 × coating buffer before coating
Albumen after dissolved dilution is diluted to corresponding peridium concentration, is sub-packed in the Eppendorf pipe of 20ml.
Coating: the albumen coating buffer prepared in advance is mixed well, pours into clean loading slot, uses Multi-channel liquid transfer device
It sequentially adds in 96 hole elisa Plates, every 50 μ l of hole;After every plate sample-adding, jiggles uniformly, guarantee that coating buffer is laid in often
Hole bottom;" protein name+group " is marked on ELISA Plate, is covered with preservative film, ELISA Plate is then horizontally placed at 4 DEG C of mistakes
Night.
Closing: the ELISA Plate after taking out overnight abandons albumen coating buffer unadsorbed to the greatest extent, takes 100 μ l with Multi-channel liquid transfer device
Block buffer (BSA) adds in each hole of ELISA Plate, then covers ELISA Plate with preservative film, is placed in 37 DEG C of thermostat water baths,
Successively record standing time, water-bath 2h;After the water bath is over, ELISA Plate is taken out, Block buffer unbonded to the greatest extent is abandoned;Board-washing
After machine completes auto-flushing pipeline, automatic washer parameter is set are as follows: 350 μ l of dcq buffer liquid, two o'clock imbibition impregnate, wash 3
Time;It is sequentially placed into ELISA Plate and carries out board-washing, after cleaning, successively patted dry on blotting paper.
Primary antibody is incubated for: the serum diluted in advance taken out, the serum after mixing dilution is sufficiently blown and beaten with Multi-channel liquid transfer device,
Then 50 μ l are drawn, 96 hole elisa Plates are sequentially added;The universal antibody dilution of equivalent is only added in blank well, it is negative and positive
Control wells are separately added into the yin-yang serum after dilution in proportion;Sample-adding finishes, and is sequentially placed into 37 DEG C of thermostat water baths, records respectively
Time;After water-bath 1h, board-washing 3 times, pat dry.
Secondary antibody is incubated for: being taken out ELISA Plate, is discarded liquid extra in hole.HRP-Rec-A is pressed with universal antibody dilution
1:4000 dilution is drawn 50ul with Multi-channel liquid transfer device, is sequentially added in 96 hole elisa Plates;Sample-adding finishes, and successively puts 37 DEG C of perseverances
Warm water bath, records the time respectively;Water-bath 1h board-washing 3 times, is patted dry.
Colour developing: the reaction solution and solution A in colour reagent box are taken out, under the conditions of being protected from light, according to reaction solution 10ml+ solution A
The preparation method of 2.5 μ l prepares developing solution, is protected from light and is placed in a beaker after mixing well;Appropriate developing solution is taken to pour into clean sample-adding
In slot, 50 μ l are drawn with multi-pass amount pipettor, are sequentially added in 96 hole elisa Plates;Sample-adding finishes, and is protected from light and is placed in 37 DEG C of thermostatted waters
Bath records the time respectively;After water-bath 15min, every hole sequentially adds 25 μ l terminate liquids;ELISA Plate bottom is dried with blotting paper.
Upper machine: setting microplate reader photometric measurement wavelength 405nm in advance;After terminate liquid is added in ELISA Plate, machine testing is gone up immediately,
And save testing result.
4. statistical analysis technique
The present invention be respectively adopted first two independent samples t tests and Mann-Whiteny U inspection compare test phase lung cancer group and
Whether the mean of autoantibody and median are variant in Normal group;One-way analysis of variance and Kruskal- is respectively adopted
Wallis examine compare Qualify Phase lung cancer group, the mean of autoantibody and median are in benign tuberculosis group and Normal group
It is no variant;Youden index maximum OD value when ROC curve determines the judgment criteria of antibody positive, i.e. 90% or more specificity;Root
Judge antibody to the distinguishing ability of lung cancer according to area under the curve (AUC).All statistical analysis are carried out using SPSS21.0 software, and P <
0.05 is Statistic analysis standard.
5. clinically application of the detection anti-MM P12 autoantibody in pulmonary cancer diagnosis
1) distinguishing ability of the anti-MM P12 autoantibody to lung cancer and normal person in test group
184 Serum of Patients with Lung Cancer are measured by ELISA and 184 normal control serum carry out anti-MM P12 autoantibody
Detection, Serum of Patients with Lung Cancer antibody titer is apparently higher than Normal group (Fig. 2) as the result is shown, anti-MM P12 autoantibody lung
Cancer is 0.7 for the AUC of normal person, and sensitivity and specificity are respectively 20.70% and 90.80%, and P value is respectively less than 0.001(figure
3).Lung cancer and normal person can be distinguished by present invention prompt, anti-MM P12 autoantibody well.
2) anti-MM P12 autoantibody is worth lung cancer detection in validation group
Present invention ELISA detection method is to 446 patients with lung cancer, 119 benign consumptives and 446 Sex, Ages
The normal human serum matched carries out the detection of anti-MM P12 autoantibody, the results show that anti-MM P12 in Serum of Patients with Lung Cancer
Autoantibody is significantly higher than benign consumptive and normal control (Fig. 4).ROC is analyzed as the result is shown (such as Fig. 5), lung cancer pair
In the area under the curve AUC of normal person and credibility interval be 0.64(95%CI:0.608-0.680,P< 0.001), sensitivity and spy
Different degree is respectively 25.11% and 90.13%;Lung cancer is 0.68(95% for the area under the curve AUC of benign tuberculosis and credibility interval
CI:0.627-0.732,P< 0.001);In addition, anti-MM P12 is certainly compared with non-lung cancer group (benign tuberculosis group+Normal group)
The AUC of body antibody is 0.601(95%CI:0.567-0.636,P< 0.001).Result prompt of the present invention, anti-MM P12 autoantibody
It can be used as the biomarker of lung cancer detection.
6. the relationship of anti-MM P12 autoantibody and patients with lung cancer Clinical symptoms
In the patients with lung cancer of Qualify Phase, there are 160 patients that there is detailed information by stages, regard I phase and II as early stage lung
Cancer group, III phase and IV phase are as advanced lung cancer group.By different sexes, age grouping, clinical stages, histological type, whether there is or not transfer,
Whether there is or not the autoantibodies rates of the patients with lung cancer of Smoking And Drinking history to be compared analysis, and the results are shown in Table 2.Anti-MM P12 is certainly
Body antibody the positive rate in old group (>=60 years old) be apparently higher than in non-aged group (<60 years old) positive rate (P< 0.05).
Anti-MM P12 autoantibodies rate in 2 patients with lung cancer of table
Claims (5)
1. a kind of lung cancer marker, which is characterized in that the lung cancer marker is anti-MM P12 autoantibody.
2. a kind of lung cancer marker anti-MM P12 autoantibody as described in claim 1 is used for lung cancer detection kit in preparation
In application.
3. detecting the kit of lung cancer according to claim 2, which is characterized in that the kit is inhaled based on enzyme linked immunological
The detection architecture of adhesion test.
4. detecting the kit of lung cancer according to claim 3, which is characterized in that the kit contains detection anti-MM P12
The MMP12 of autoantibody recombinates purifying protein.
5. detecting the kit of lung cancer according to claim 3, which is characterized in that the kit also contains ELISA coating
Buffer, Block buffer, HRP-Rec-A secondary antibody, ELISA universal antibody dilution and ELISA-ABTS colour reagent, PBST,
Terminate liquid.
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