CN103848890B - The antigenic peptide of MUC1 autoantibody identification - Google Patents
The antigenic peptide of MUC1 autoantibody identification Download PDFInfo
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Abstract
The present invention relates to the antigenic peptide of MUC1 autoantibody identification.Wherein, this polypeptide to have as shown in SEQ ID NO:1-5 aminoacid sequence one of at least.Utilize the antigenic peptide of MUC1 autoantibody of the present invention identification, effectively can detect sample, such as, MUC1 autoantibody in the blood sample (such as serum sample) of lung cancer or doubtful patients with lung cancer.
Description
Technical field
The present invention relates to biomedical sector.Particularly, the present invention relates to antigenic peptide of MUC1 autoantibody identification and uses thereof.More specifically, the present invention relates to the antigenic peptide of a kind of MUC1 autoantibody identification, this polypeptide detect tumour serum MUC1 antibody purposes, a kind of test kit for detecting MUC1 antibody in sample in preparation.
Background technology
Lung Cancer is very complicated, grade malignancy is high and by force invisible, and the key factor of high mortality is to lack effective early diagnosis.As everyone knows, the prognosis of lung cancer and the by stages closely related of this disease during diagnosis.Patients with lung cancer is in different neoplasm stagings, and its 5 annual survival rate is as follows: I phase 5 annual survival rate 60-70%, II phase 5 annual survival rate 40-50%, and IIIB phase and IV phase are 5-20%.Major part lung cancer is do not have clinical symptom in the painless phase (about 8 years), simultaneously, effective screening method is not still had to find the early stage of lung cancer clinically, clinical conventional several lung cancer marker, as neuronspecific enolase (NSE), gastrin-releasing peptide precursor (pro-GRP), Cyfra21-1 (CYFRA21-1), p53 antibody and carcinomebryonic antigen (CEA) etc., also neither one tumor markers finds that there is enough susceptibility, specificity and repeatability to the early stage of lung cancer, therefore constrains us and carries out Effective selection to the early stage of lung cancer.Therefore, we think in the urgent need to finding effective tumor related marker thing for lung cancer early diagnosis.
Tumour autoantibody cancerates in process in tumour, because new protein produces, some protein is overexpressed simultaneously, thus the immunne response of excitating organism, produce the multiple antibody for autoantigen and tumour antigen, although its generation mechanism and effect are also unclear, the existence of these autoantibodies shows to play a role in humoral immunization identification tumour antigen.Compared to serum tumor antigenic type mark, it is high that the corresponding autoantibody of serum tumor antigen has abundance, occurs that early, the advantages such as easy detection, may become more reliable blood serum tumor markers.
MUC1 is that a kind of film mating type is mucoprotein, has abundant expression, play lubrication and provide protection to normal epithelium at the surface epithelial cell of many tissues; Meanwhile, MUC1 can interact with different kinds of molecules, thus affects the biochemical functions of cell.The unconventionality expression of MUC1 is also a remarkable characteristic of kinds cancer, and due to MUC1 unconventionality expression in kinds of tumors, mainly comprising (1) expression amount increases, more than 100 times when can reach normal; (2) change of cell surface distribution, polar contribution is lost, and whole cell surface is all expressed; (3) structural modification, incomplete mainly due to glycosylation, there is Aberrant glycosylation and aberrant polypeptide epi-position, become a kind of potential tumor related marker thing.For the feature of its unconventionality expression in tumour cell, infer that the detection of himself antibody expression more may can reflect this change.But at present for the detection of MUC1 autoantibody, mostly use MUC1 full-length proteins, and the preparation of total length pure protein and purge process being comparatively complicated, difficulty is comparatively large, considerably increases testing cost; And during full-length proteins detection autoantibody, epi-position exposes to the open air not good, makes the Sensitivity and Specificity of detection all undesirable, therefore, find more preferably antigen epitope polypeptide and replace full-length proteins to become an effective solution route undoubtedly.
Summary of the invention
The present invention is intended to solve MUC1 pure protein expression in existing MUC1 autoantibody detection kit, purifying difficulty is high, the defect that test kit cost is high, detection sensitivity is low, and provides a kind of useful business to select.
Another object of the present invention is to provide the application of the antigenic peptide of above-mentioned MUC1 autoantibody identification in preparation lesion detection test kit.
Another object of the present invention is to provide the antigenic peptide of above-mentioned MUC1 autoantibody identification to detect tumour MUC1 autoantibody test kit.
For this reason, the present invention proposes the antigenic peptide method how obtaining the identification of effective MUC1 autoantibody, and further provide the means that effectively can detect MUC1 autoantibody in sample.
Thus, the present invention proposes the antigenic peptide of a kind of MUC1 autoantibody identification.According to embodiments of the invention, its polypeptide active fragment be following one of at least:
1) sequence: SFFFLSFHISNLQFNSSLED shown in SEQ ID No:1;
2) sequence: NLQFNSSLEDPSTDYYQELQ shown in SEQ ID No:2;
3) sequence: TAPPVHNVTSASGSASGSAS shown in SEQ ID No:3;
4) sequence: PFSIPSHHSDTPTTLASHST shown in SEQ ID No:4;
5) sequence: TPTTLASHSTKTDASSTHHS shown in SEQ ID No:5;
6) aminoacid sequence shown in SEQ ID No:1-5 is through the replacement of amino-acid residue, disappearance or interpolation and the amino acid derived sequence formed.
According to embodiments of the invention, preferably, the antigenic peptide of MUC1 autoantibody of the present invention identification has as SFFFLSFHISNLQFNSSLED(SEQ ID NO:1) shown in aminoacid sequence.Thus, the efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.In addition, the invention allows for the purposes of antigenic peptide in preparation detection tumour serum MUC1 antibody kit of foregoing MUC1 autoantibody identification.Especially, the tumour that this test kit may be used for detecting is lung cancer.
For this reason, the present invention proposes a kind of test kit for detecting MUC1 antibody in sample.According to embodiments of the invention, this test kit comprises: the antigenic peptide of foregoing MUC1 autoantibody identification.Particularly, according to embodiments of the invention, this test kit can comprise: 96 orifice plates, wraps by the antigenic peptide of foregoing MUC1 autoantibody identification in the hole of described 96 orifice plates.Further, this test kit may further include: negative control sample, described negative control sample is MUC1 antibody is negative serum, or/may further include extraly: standard model, described standard model is that MUC1 antibody concentration is respectively 1U/ml, the serum of 5U/ml, 10U/ml, 15U/ml.According to embodiments of the invention, the concentration of the antigenic peptide of MUC1 autoantibody identification can be each reacting hole 1 μ g/ml.According to embodiments of the invention, especially, the tumour that this test kit may be used for detecting is lung cancer.
For this reason, in still another aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, this polypeptide to have as shown in SEQ ID NO:1-5 aminoacid sequence one of at least.That is, polypeptide has as one of following shown aminoacid sequence:
SFFFLSFHISNLQFNSSLED（SEQ ID NO:1）
NLQFNSSLEDPSTDYYQELQ（SEQ ID NO:2）
TAPPVHNVTSASGSASGSAS（SEQ ID NO:3）
PFSIPSHHSDTPTTLASHST（SEQ ID NO:4）
TPTTLASHSTKTDASSTHHS（SEQ ID NO:5）。
According to embodiments of the invention, utilize the polypeptide with above-mentioned aminoacid sequence, effectively can detect sample, such as, MUC1 autoantibody in the blood sample (such as serum sample) of lung cancer or doubtful patients with lung cancer.
According to embodiments of the invention, preferably described polypeptide has as SFFFLSFHISNLQFNSSLED(SEQ ID NO:1) shown in aminoacid sequence.Thus, the efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.
In still another aspect of the invention, the present invention proposes polypeptide noted earlier in the purposes detecting MUC1 autoantibody in sample.
According to embodiments of the invention, described sample is the blood sample from lung cancer or doubtful patients with lung cancer and healthy volunteer.
In still another aspect of the invention, the present invention proposes polypeptide noted earlier and preparing the purposes in test kit, described test kit is for detecting the MUC1 autoantibody in sample.
According to embodiments of the invention, described sample is the blood sample from lung cancer or doubtful patients with lung cancer and healthy volunteer.
In still another aspect of the invention, the present invention proposes a kind of test kit for detecting MUC1 autoantibody in sample.According to embodiments of the invention, this test kit comprises: foregoing polypeptide.Thus, utilize according to embodiments of the invention, can effectively detect the MUC1 autoantibody in sample by polypeptide according to the present invention.
According to embodiments of the invention, this test kit may further include: be suitable for the reagent carrying out ELISA detection.Thus, ELISA detection method can be passed through, utilize polypeptide according to the present invention to detect the MUC1 autoantibody in sample, thus the efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.
According to embodiments of the invention, described polypeptide is arranged in 96 orifice plates.Thus, can detect easily high-throughput.The efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.
According to embodiments of the invention, described sample is the blood sample from lung cancer or doubtful patients with lung cancer and healthy volunteer.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Embodiment
Be described below in detail embodiments of the invention, described embodiment is intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.The antigenic peptide of MUC1 autoantibody identification
According to an aspect of the present invention, the present invention proposes the antigenic peptide of a kind of MUC1 autoantibody identification.According to embodiments of the invention, its polypeptide active fragment be following one of at least:
1) sequence: SFFFLSFHISNLQFNSSLED shown in SEQ ID No:1;
2) sequence: NLQFNSSLEDPSTDYYQELQ shown in SEQ ID No:2;
3) sequence: TAPPVHNVTSASGSASGSAS shown in SEQ ID No:3;
4) sequence: PFSIPSHHSDTPTTLASHST shown in SEQ ID No:4;
5) sequence: TPTTLASHSTKTDASSTHHS shown in SEQ ID No:5;
6) aminoacid sequence shown in SEQ ID No:1-5 is through the replacement of amino-acid residue, disappearance or interpolation and the amino acid derived sequence formed.
According to embodiments of the invention, preferably, the antigenic peptide of MUC1 autoantibody of the present invention identification has as SFFFLSFHISNLQFNSSLED(SEQ ID NO:1) shown in aminoacid sequence.Thus, the efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.
In another aspect of the invention, the present invention proposes a kind of isolated polypeptide.According to embodiments of the invention, this polypeptide to have as shown in SEQ ID NO:1-5 aminoacid sequence one of at least.That is, polypeptide has as one of following shown aminoacid sequence:
SFFFLSFHISNLQFNSSLED（SEQ ID NO:1）
NLQFNSSLEDPSTDYYQELQ（SEQ ID NO:2）
TAPPVHNVTSASGSASGSAS（SEQ ID NO:3）
PFSIPSHHSDTPTTLASHST（SEQ ID NO:4）
TPTTLASHSTKTDASSTHHS（SEQ ID NO:5）。
The corresponding relation of these polypeptide and MUC1 total length is as shown in the table:
It should be noted that, position listed in upper table is and (full length sequence of MUC1 is shown in NCBI NP 001191214) corresponding to the full length sequence of MUC1.In addition, term " polypeptide " used in this article refers to that at least two amino acid are connected by peptide bond and the oligomer obtained, and wherein, the number of the amino-acid residue comprised in polypeptide is also not particularly limited.According to embodiments of the invention, polypeptide can contain 20 amino acid.Certainly, it will be appreciated by persons skilled in the art that and can also carry out chemically modified to aforementioned polypeptides, to increase the antigenicity of polypeptide, contribute to polypeptide bag by the bottom of elisa plate.According to embodiments of the invention, utilize the polypeptide with above-mentioned aminoacid sequence, effectively can detect sample, such as, MUC1 autoantibody in the blood sample (such as serum sample) of lung cancer or doubtful patients with lung cancer.Contriver finds that the aforementioned polypeptides obtained from MUC1 complete sequence can as the epitope of MUC1, can with the reaction of MUC1 autoantibody specificity in blood sample, thus, can effectively for the MUC1 autoantibody in detected object blood sample, thus, can effectively for examination lung cancer suspected patient serum, the diagnosis for lung cancer provides a new test rating.
According to embodiments of the invention, preferably described polypeptide has as SFFFLSFHISNLQFNSSLED(SEQ ID NO:1) shown in aminoacid sequence.Thus, the efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.According to embodiments of the invention, contriver finds, anti-MUC1
261-280the susceptibility of (SEQ ID NO:1) autoantibody in patients with lung cancer diagnosis is 29.2%, and in lung cancer (I-II phase), susceptibility is 38% in early days, and specificity is 97.4%.Thus, examination lung cancer suspected patient serum special polypeptide of the present invention is shown, the specific polypeptides MUC1 of especially MUC1 autoantibody identification
261-280(SEQ ID NO:1), can be used for the auxiliary diagnosis of lung cancer, especially the early screening of lung cancer.
It will be appreciated by persons skilled in the art that can effectively by conventional synthesis process, synthesis aforementioned polypeptides, thus replaces recombinant expressed biosynthesizing mode.
The application of the antigenic peptide of MUC1 autoantibody identification and test kit
Thus, in another aspect of the invention, the invention allows for polypeptide noted earlier in the purposes detecting MUC1 autoantibody in sample.According to embodiments of the invention, can the type of the sample detected be carried out by aforementioned polypeptides and be not particularly limited.According to embodiments of the invention, preferred sample is the blood sample from lung cancer or doubtful patients with lung cancer.Thus, the MUC1 autoantibody in the blood sample of polypeptide detection of lung cancer or doubtful patients with lung cancer can be utilized, can determine whether patient suffers from malignant tumour thus, provide supplementary means for malignant tumour detects, or the early screening of malignant tumour such as lung cancer is provided.
Polypeptide of the present invention is utilized to detect the method for MUC1 autoantibody in sample and be not particularly limited.According to embodiments of the invention, can enzyme linked immunosorbent assay (ELISA) method be passed through, detect the MUC1 autoantibody in sample by polypeptide of the present invention.Thus, the efficiency of the MUC1 autoantibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.Exemplarily, following method can be adopted to utilize MUC1 antibody in the blood sample of polypeptide detection of lung cancer or doubtful patients with lung cancer:
First, with the carbonate buffer solution (NaCO of pH9.6
30.159g, NaHCO
30.293g, adds water to 100ml, and pH value is 9.6) dilution polypeptide to 1 μ g/ml, join in the hole of 96 hole enzyme plates with the amount in 100 μ l/ holes, after shrouding film phonograph seal, 4 DEG C are spent the night.
Next, discard the liquid in hole, 0.1%TBS-T lavation buffer solution is (namely containing the TBS-T solution of 0.1%Tween-20, its compound method is: NaCl 8.7g, Tris 1.21g, adds deionized water to 1000ml, pH 7.5, then add Tween-201ml) detersive enzyme target 5 times, each 3min.
Then, (its compound method is Xiang Kongzhong interpolation 5%BSA/0.05%TBS-T Block buffer: NaCl0.87g, Tris 0.121g, add deionized water to 100ml, pH 7.5, then add Tween-200.5ml, BSA 5g), every hole 300 μ l, hatches 1h for 37 DEG C after shrouding film phonograph seal.
Then, testing sample is added.With 5%BSA/0.05%TBS-T Block buffer dilute serum, Dilution ratio is 1:100, discard liquid in hole completely, every hole increase serum diluent 100 μ l, the multiple hole of every routine serum sample 3,2 Positive control wells set up by every plate, add known positive serum, 2 blank control wells set up by every plate, add 0.1%TBS-T lavation buffer solution.2h is hatched for 37 DEG C after shrouding film phonograph seal.Discard liquid in hole, detersive enzyme target 5 times, each 3min.
Then, second antibody immune response is carried out.Utilize 5%BSA/0.05%TBS-T Block buffer to dilute the Goat anti human IgG of horseradish peroxidase-labeled, Dilution ratio 1:5000, every hole adds 100 μ l, hatches 1h for 37 DEG C after shrouding film phonograph seal.Discard liquid in hole, detersive enzyme target 5 times, each 3min.
Then, substrate colour developing is added.Conveniently ELISA kit specification sheets (such as health is century TMB colouring reagents box), add chromogenic substrate, every hole adds 100 μ l, and develop the color under room temperature lucifuge condition 10min, and every hole adds 50 μ l 2M H
2sO
4termination reaction.
Then, optical density(OD) OD value is measured under microplate reader 492nm wavelength condition.
Finally, based on the difference of obtained optical density(OD) OD value and predetermined reference value, determine whether testing sample comes from and suffer from patients with lung cancer.According to embodiments of the invention, the blood sample making a definite diagnosis cancer patients and Healthy People (healthy volunteer) can be utilized to carry out numerical value that parallel test obtains is as predetermined reference value.
Such as, the autoantibody of the anti-MUC1 in blood sample is detected by ELISA method.If the optical density value of the chromogenic enzyme substrate reaction in sample to be tested is higher than X+2SD, then test serum is lung cancer suspected patient serum, wherein in X+2SD, X is the OD average of the chromogenic enzyme substrate reaction in Healthy Human Serum, and 2SD is two times of standard deviations of the optical density value that the chromogenic enzyme substrate in Healthy Human Serum reacts.Particularly, according to embodiments of the invention, X+2SD value of the present invention is obtained by following method: according to the aforementioned method utilizing polypeptide of the present invention to detect MUC1 autoantibody in sample, adopt 400 routine normal healthy controls blood samples, measure the OD value of its chromogenic enzyme substrate reaction, after the OD value recorded is met normal distribution (p>0.05) with single sample K-S check analysis, calculate and obtain the mean value (X=0.33) of each OD value and standard deviation (SD=0.13), then with X+2SD(0.59) be the upper limit of normal value.
According to a further aspect in the invention, the invention allows for the purposes of antigenic peptide in preparation detection tumour serum MUC1 antibody kit of foregoing MUC1 autoantibody identification.Especially, the tumour that this test kit may be used for detecting is lung cancer.
For this reason, in accordance with a further aspect of the present invention, the present invention proposes a kind of test kit for detecting MUC1 antibody in sample.According to embodiments of the invention, this test kit comprises: the antigenic peptide of foregoing MUC1 autoantibody identification.Particularly, according to embodiments of the invention, this test kit can comprise: 96 orifice plates, wraps by the antigenic peptide of foregoing MUC1 autoantibody identification in the hole of described 96 orifice plates.Further, this test kit may further include: negative control sample, described negative control sample is MUC1 antibody is negative serum, or/may further include extraly: standard model, described standard model is that MUC1 antibody concentration is respectively 1U/ml, the serum of 5U/ml, 10U/ml, 15U/ml.According to embodiments of the invention, the concentration of the antigenic peptide of MUC1 autoantibody identification can be, each reacting hole 1 μ g/ml.According to embodiments of the invention, especially, the tumour that this test kit may be used for detecting is lung cancer.
In still another aspect of the invention, the present invention proposes polypeptide noted earlier and preparing the purposes in test kit, described test kit is for detecting the MUC1 autoantibody in sample.According to embodiments of the invention, described sample is the blood sample from lung cancer or doubtful patients with lung cancer.Thus, the MUC1 autoantibody in the blood sample of polypeptide detection of lung cancer or doubtful patients with lung cancer can be utilized, can determine whether patient suffers from malignant tumour thus, provide supplementary means for malignant tumour detects, or the progress of monitoring malignant tumour such as lung cancer.
In another aspect of this invention, the present invention proposes a kind of test kit for detecting MUC1 autoantibody in sample.According to embodiments of the invention, this test kit comprises: foregoing polypeptide.Thus, utilize according to embodiments of the invention, can effectively detect the MUC1 antibody in sample by polypeptide according to the present invention.According to embodiments of the invention, this test kit may further include: be suitable for the reagent carrying out ELISA detection.Thus, can ELISA detection method be passed through, utilize polypeptide according to the present invention to detect the MUC1 antibody in sample, thus the efficiency of the MUC1 antibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.According to embodiments of the invention, described polypeptide is arranged in 96 orifice plates, such as, can wrap by the hole of 96 orifice plates, such as can by conventional processing as undertaken by coating buffer and retardance liquid.Thus, can detect easily high-throughput.The efficiency of the MUC1 antibody in the blood sample utilizing polypeptide detection of lung cancer or doubtful patients with lung cancer can be improved further.Can also comprise other reagent in test kit, such as dcq buffer liquid, sample dilution buffer, tmb substrate solution, reaction terminating liquid, two of horseradish enzyme labelling resists, negative control sample, standard model.Wherein, negative control sample is MUC1 antibody is negative serum, and standard model is that MUC1 antibody concentration is respectively 1U/ml, the serum of 5U/ml, 10U/ml, 15U/ml.
According to embodiments of the invention, described sample is the blood sample from lung cancer or doubtful patients with lung cancer.
It should be noted that, in this article, sometimes also by " antigenic peptide of MUC1 autoantibody identification " referred to as " polypeptide ", by " MUC1 autoantibody " referred to as " MUC1 antibody ".
Below by specific embodiment, the present invention is made an explanation.It should be noted that, the following example is only illustrative, and does not limit the present invention in any way.In addition, all appts adopted in the following example, material etc. are commercially available, as the operation do not explicitly not pointed out in the examples below that, can be undertaken by the working method of those skilled in the art's routine.
The preparation of embodiment 1 polypeptide
1.1 general method
According to the full length amino acid residue sequence (NP 001191214) of MUC1 albumen, adopt situ synthesis techniques to be the polypeptide chip (Piptide Array) of the polypeptide of 20 at activated cellulose film surface composition length, each polypeptide is overlapping 10 amino-acid residues of order directly.MUC1 full-length proteins is containing 475 amino-acid residues, and MUC1 polypeptide array comprises 20 galanin peptide points of overlapping 10 aminoacid sequences.
1.2 fabricated in situ prepare polypeptide chip (Piptide Array)
Peptide synthesizer ASP SL(Germany is carried out according to albumen and polypeptide amino acid residues in length, overlap length, intavis company) setting of MutilPep synthesis sequence of control, add the nitrocellulose filter surface synthesis site of a kind of specific amino acids to activation according to time variable control at each synthesis cycle, and between catalytic amino acid, peptide bond is formed.Synthetic peptide chain length is 20 amino-acid residues, need carry out the reaction in 20 cycles, according to the operating process of ASP SL Peptide synthesizer, the native amino acid solution of 20 kinds of FMOC-radical protections is put into position corresponding on machine.Between each cycle, amino acid will through capping, and a series of processes such as FMOC-group deprotection could be combined with next amino acid, during last end cycle, amino acid whose side chain protected group be taken off.The steps include: that the amino acid of specifying is added drop-wise to specific position on nitrocellulose filter by Peptide synthesizer ASP SL, the solution of acetic anhydride of 2% washes film 5 minutes, and dimethyl formamide washes film 5 times, each 2 minutes.The piperidine solution of 20% is washed film and is sloughed FMOC-blocking group in 10 minutes, and dimethyl formamide washes film 5 times, each 2 minutes.Ethanol washes film 2 times, each 2 minutes, dries.Repeat aforesaid operations, until complete 20 cycles, during last end cycle, the piperidine solution of 20% washes film 2 times, each 10 minutes.Dimethyl formamide washes film 5 times, each 2 minutes.Air dried overnight after film 2 times washed by ethanol.Finally, 10ml trifluoroacetic acid, 300 μ l methylene dichloride, 200 μ l water fully mix, and wash film 1 hour, slough side chain protected group with mixed solution, and methylene dichloride washes film 5 times, each 2 minutes.Dimethyl formamide washes film 5 times, each 2 minutes.Ethanol dries after washing film 2 times.Save backup-20 DEG C of sealings after polypeptide chip synthesis.
Embodiment 2 screens the polypeptide relevant to lung cancer
The aquation of 2.1 polypeptide chips
First, the polypeptide chip prepared by embodiment 1 is immersed in 40ml 100% ethanol, shaking table shakes 5 minutes.Then, this polypeptide chip is immersed in 40ml 75% ethanol, shaking table shakes 5 minutes.Next, polypeptide chip is immersed in 40ml 50% ethanol, shaking table shakes 5 minutes.Then, add 150ml PBS to soak 30 minutes (fade completely with the white point on film and be as the criterion).Finally, just polypeptide chip immerses in 40ml 5% skim-milk/PBS-T solution, and incubated at room temperature is closed for 3 hours.
2.2 polypeptide array and sero-reaction
By 10 routine Serum of Patients with Lung Cancer balanced mix, prepare Sera of Lung Cancer pond, immune response liquid is prepared into 5% skim-milk/PBS-T solution 1: 1000 dilution, polypeptide chip is put into hybridization bag, reaction solution is added by 0.1ml/cm2, remove all bubbles in bag, film sealing machine seals, 4 DEG C of shaken overnight gently.
By 10 routine normal healthy controls serum mixing, preparation normal healthy controls serum pond, is prepared into immune response liquid with 5% skim-milk/PBS-T solution 1:1000 dilution, polypeptide chip is put into hybridization bag, adds reaction solution by 0.1ml/cm2, remove all bubbles in bag, film sealing machine seals, 4 DEG C of shaken overnight gently.
After seroimmunity reaction terminates, cut off hybridization bag, discard reaction solution, PBS-T 40ml washes polypeptide chip 6 times, each 10 minutes, polypeptide chip is put into hybridization bag, adds the Goat anti human IgG solution of 1:10000 dilution, by 0.1ml/cm
2add two anti-reaction solutions, seal hybridization bag, room temperature with gentle vibrates 2 hours, and 40ml PBS-T washes film 3 times, each 10 minutes, and 40ml PBS washes film 3 times, each 10 minutes.
Next, carry out ECL development, exposure, scans film, with AlphaView software system analysis image.
2.3 screening
According to above-mentioned Piptide Array(polypeptide chip) immune response result, select with Serum of Patients with Lung Cancer reacting positive and differential peptides point (sequence in table 1) seronegative with normal healthy controls group, composition polypeptide original position Dot-Blot film, according to preparation method described in embodiment 1, preparation comprises these 5 polypeptide and to impinging upon interior polypeptide chip, calculating the positive frequency value of each point by carrying out immune response with 29 routine Serum of Patients with Lung Cancers, determining specific sequence (seeing the following form 1).As shown in table 1, wherein, that susceptibility is the highest is MUC1
261-280(SEQ ID NO:1).
Table 1
Embodiment 3 determines the upper limit of the anti-MUC1 specific polypeptides autoantibody normal value of normal healthy controls serum
The upper limit by the autoantibody normal value of anti-MUC1 specific polypeptides in following method determination normal healthy controls serum:
First, the specific polypeptides MUC1 of the MUC1 autoantibody identification obtained is screened with embodiment 2
261-280as detectable antigens, utilize the MUC1 autoantibody in ELISA method detection normal healthy controls (n=400) serum, concrete grammar is as follows:
First, MUC1 is synthesized with Peptide synthesizer MultiPep RS according to amino-acid residue order in sequence table
261-280, and by obtained peptide coupling KLH, to increase the antigenicity of polypeptide, contribute to polypeptide bag by the bottom of elisa plate.
Bag quilt: with the carbonate buffer solution (NaCO of pH9.6
30.159g, NaHCO
30.293g, adds water to 100ml, and pH value is 9.6) dilute the MUC1 synthesized
261-280to 1 μ g/ml, add 96 hole enzyme plates with 100 μ l/well, after shrouding film phonograph seal, 4 DEG C are spent the night.
Washing: discard the liquid in hole, 0.1%TBS-T lavation buffer solution (its compound method is: NaCl 8.7g, Tris 1.21g, adds deionized water to 1000ml, pH 7.5, then adds Tween-201ml) detersive enzyme target 5 times, each 3min.
Close: (its compound method is 5%BSA/0.05%TBS-T Block buffer: NaCl 0.87g, Tris0.121g, adds deionized water to 100ml, pH 7.5, then add Tween-20 0.5ml, BSA 5g), every hole 300 μ l, hatches 1h for 37 DEG C after shrouding film phonograph seal.
Add serum to be checked: with 5%BSA/0.05%TBS-T Block buffer dilute serum, Dilution ratio is 1:100, discard liquid in hole completely, every hole increase serum diluent 100 μ l, the multiple hole of every routine serum sample 3,2 Positive control wells set up by every plate, add known positive serum, 2 blank control wells set up by every plate, add 0.1%TBS-T lavation buffer solution.2h is hatched for 37 DEG C after shrouding film phonograph seal.Discard liquid in hole, detersive enzyme target 5 times, each 3min.
Second antibody immune response: the Goat anti human IgG of 5%BSA/0.05%TBS-T Block buffer dilution horseradish peroxidase-labeled, Dilution ratio 1:5000, every hole adds 100 μ l, hatches 1h for 37 DEG C after shrouding film phonograph seal.Discard liquid in hole, detersive enzyme target 5 times, each 3min.
Add substrate colour developing: use TMB colouring reagents box, the specification sheets provided according to institute of manufacturers (health is century), develop the color under room temperature lucifuge condition 10min, and every hole adds 50 μ l 2M H
2sO
4termination reaction.
Detect: under microplate reader 492nm wavelength condition, measure optical density(OD) OD value.
Then, the above-mentioned OD value recorded is met after normal distribution (p>0.05) through single sample K-S check analysis, calculate mean value X and the standard deviation SD of each OD value, represent the upper limit of the autoantibody normal value of anti-MUC1 specific polypeptides in normal healthy controls serum with X+2SD.As a result, the mean value X=0.33 of each OD value, standard deviation SD=0.13, then in normal healthy controls serum, the upper limit X+2SD of the autoantibody normal value of anti-MUC1 specific polypeptides is 0.59.
Embodiment 4 polypeptide compares for the positive rate detecting MUC1 autoantibody in lung good foul disease patient serum
In the present embodiment, according to the ELISA detection method described in embodiment 3, screen the MUC1 obtained with embodiment 2
261-280(SEQ ID NO:1) specific polypeptide as antigen, the anti-MUC1 in detection of lung cancer patient (n=406) serum, lung benign disease patient (n=66) serum and normal healthy controls (n=89) serum
261-280autoantibody, and to utilizing the positive rate of polypeptide detection of lung cancer and multiple Benign Pulmonary Nodules Disease serum MUC1 autoantibody to analyze.The results are shown in following table 2-5.Wherein, when the upper limit 0.59 of the autoantibody normal value of anti-MUC1 specific polypeptides during the OD value that the chromogenic enzyme substrate in serum sample reacts is higher than embodiment 3 determined normal healthy controls serum, this serum is lung cancer suspected patient serum.
Wherein, the MUC1 in each serum sample
261-280autoantibodies rate is as shown in table 2 below:
Table 2
| Sample type | Number of cases | MUC1 261-280Antibody positive rate |
| Lung cancer | 406 | 118/406(29.2%)* |
| Tuberculosis | 24 | 0/24(0%)* |
| Pneumonia and propping up slowly | 23 | 0/23(0%)* |
| Pulmonary abscess | 19 | 0/19(0%)* |
| Normal healthy controls | 89 | 4/89(4.5%)* |
Note: *, the difference of lung cancer group and non-lung cancer group is p<0.01
Above-mentioned relatively in, lung cancer group case load is 406, and the case load of non-lung cancer group (comprising lung's benign disease and normal healthy controls) is 155 examples, anti-MUC1 in lung cancer group and non-lung cancer group serum
261-280autoantibodies and negative number of cases list in table 3 respectively.
Table 3
| MUC1 autoantibody | Patients with lung cancer number | Non-lung cancer patient number |
| Positive | 118(a, i.e. true positives) | 4(b, i.e. false positive) |
| Negative | 288(c, i.e. false negative) | 151(d, i.e. true negative) |
Then, anti-MUC1 is calculated
261-280the Sensitivity and Specificity of autoantibody in patients with lung cancer diagnosis, calculation formula is as follows:
Susceptibility=(a/a+c) × 100%
Specificity=(d/b+d) × 100%
Positive predictive value=(a/a+b) × 100%
Negative predictive value=(d/c+d) × 100%
Result is summed up as table 4:
Table 4
| Autoantibody | Susceptibility | Specificity | Positive predictive value | Negative predictive value |
| MUC 1 261-280 | 29.2% | 97.4% | 96.7% | 34.4% |
Wherein, as shown in table 5 below, the positive rate of MUC1 autoantibody comparatively average positive rate (the i.e. positive rate of lung cancer shown in table 2: 29.2%) high in lung cancer group early stage patient serum.
Table 5
| Total number of cases | Positive number of cases | MUC1 261-280 positive rate | |
| Non-lung cancer group | 155 | 4 | 2.6% |
| The early stage of lung cancer (I phase, II phase) | 179 | 68 | 38%* |
Note: *, the difference p<0.001 of the early stage of lung cancer and non-lung cancer group
To sum up, contriver finds, anti-MUC1
261-280the susceptibility of (SEQ ID NO:1) autoantibody in patients with lung cancer diagnosis is 29.2%, and in lung cancer (I-II phase), susceptibility is 38% in early days, and specificity is 97.4%.Thus, show the antigenic peptide of MUC1 autoantibody of the present invention identification, the specific polypeptides MUC1 of especially MUC1 autoantibody identification
261-280(SEQ ID NO:1), can be effective to the auxiliary diagnosis of lung cancer, especially the early screening of lung cancer.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (9)
1. an antigenic peptide for MUC1 autoantibody identification, is characterized in that, its polypeptide active fragment be following one of at least:
1) sequence: SFFFLSFHISNLQFNSSLED shown in SEQ ID No:1;
2) sequence: NLQFNSSLEDPSTDYYQELQ shown in SEQ ID No:2;
3) sequence: TAPPVHNVTSASGSASGSAS shown in SEQ ID No:3;
4) sequence: PFSIPSHHSDTPTTLASHST shown in SEQ ID No:4;
5) sequence: TPTTLASHSTKTDASSTHHS shown in SEQ ID No:5.
2. the antigenic peptide of MUC1 autoantibody according to claim 1 identification, is characterized in that, the aminoacid sequence of described polypeptide is as shown in SFFFLSFHISNLQFNSSLED (SEQ ID NO:1).
3. the purposes of antigenic peptide in preparation detection of lung cancer serum MUC1 antibody kit of the MUC1 autoantibody identification described in claim 1 or 2.
4. for detecting a test kit for MUC1 antibody in sample, it is characterized in that, comprising:
The antigenic peptide of the MUC1 autoantibody identification described in claim 1 or 2.
5. test kit according to claim 4, is characterized in that, comprising:
96 orifice plates, wrap in the hole of described 96 orifice plates by the antigenic peptide of the MUC1 autoantibody identification described in claim 1 or 2.
6. test kit according to claim 5, is characterized in that, comprises further: negative control sample, and described negative control sample is MUC1 antibody is negative serum.
7. test kit according to claim 5, is characterized in that, comprises further: standard model, and described standard model is that MUC1 antibody concentration is respectively 1U/ml, the serum of 5U/ml, 10U/ml, 15U/ml.
8. test kit according to claim 5, is characterized in that, the concentration of the antigenic peptide of described MUC1 autoantibody identification is each reacting hole 1 μ g/ml.
9. test kit according to claim 4, is characterized in that, described sample is Sera of Lung Cancer.
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| WO2010138740A1 (en) * | 2009-05-27 | 2010-12-02 | Dana-Farber Cancer Institute, Inc. | Inhibition 0f inflammation using antagonists of muc1 |
| WO2011012309A1 (en) * | 2009-07-31 | 2011-02-03 | Glycotope Gmbh | Muc1 antibodies |
| CN102239182A (en) * | 2008-10-06 | 2011-11-09 | 米纳瓦生物技术公司 | Muc1* antibodies |
| CN102264765A (en) * | 2008-10-28 | 2011-11-30 | 盐野义制药株式会社 | Anti-MUC1 antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102239182A (en) * | 2008-10-06 | 2011-11-09 | 米纳瓦生物技术公司 | Muc1* antibodies |
| CN102264765A (en) * | 2008-10-28 | 2011-11-30 | 盐野义制药株式会社 | Anti-MUC1 antibody |
| WO2010138740A1 (en) * | 2009-05-27 | 2010-12-02 | Dana-Farber Cancer Institute, Inc. | Inhibition 0f inflammation using antagonists of muc1 |
| WO2011012309A1 (en) * | 2009-07-31 | 2011-02-03 | Glycotope Gmbh | Muc1 antibodies |
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