CN103304658B - Radiosensitive polypeptide mark SPG01 for nasopharynx cancer and ELISA (Enzyme-Linked Immuno Sorbent Assay) kit thereof - Google Patents

Radiosensitive polypeptide mark SPG01 for nasopharynx cancer and ELISA (Enzyme-Linked Immuno Sorbent Assay) kit thereof Download PDF

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CN103304658B
CN103304658B CN201310210634.5A CN201310210634A CN103304658B CN 103304658 B CN103304658 B CN 103304658B CN 201310210634 A CN201310210634 A CN 201310210634A CN 103304658 B CN103304658 B CN 103304658B
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spg01
nasopharyngeal carcinoma
polypeptide
synthetic peptide
antibody
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CN103304658A (en
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魏开华
夏云飞
侯利平
陶亚岚
傅海媛
杨保安
黄亚娟
宋纯艳
郑俊杰
甄蓓
张拓
韩志国
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TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
BEIJING C&N INTERNATIONAL SCI-TECH Co Ltd
Sun Yat Sen University Cancer Center
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TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
BEIJING C&N INTERNATIONAL SCI-TECH Co Ltd
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Abstract

The invention relates to a radiosensitive polypeptide mark SPG01 for nasopharynx cancer and an ELISA (Enzyme-Linked Immuno Sorbent Assay) kit thereof. The full-length amino acid sequence of the radiosensitive polypeptide mark SPG01 for nasopharynx cancer is shown as SEQ ID No.1. The polypeptide mark SPG01 provided by the invention is strong in multi-resisting specificity. The detecting specificity reaches over 90%, and the sensitivity reaches over 70%. The detection method aiming at the polypeptide mark SPG01 is simple and convenient and controllable in operation and is beneficial for clinical detection. An enzyme-linked detector can be combined for clinical detection with high throughput and low cost.

Description

A kind of nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01 and ELISA kit thereof
Technical field
The present invention relates to field of biological technology detection, specifically, relate to a kind of nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01 and ELISA kit thereof.
Background technology
Nasopharyngeal carcinoma (Nasopharyngeal carcinoma, NPC) is one of common malignant tumour of southern china, especially concentrates on Chinese Guangdong and economizes, therefore be also called " canton tumor (Canton tumor) ".Although worldwide nasopharyngeal carcinoma belongs to rare tumor, in the sickness rate of most countries or mortality ratio all lower than 1/100000, but the nasopharyngeal carcinoma annual morbidity in South China and south east asia is up to 15 ~ 50/100000, and histopathology be often diagnosed as II phase of WHO regulation or III phase (see Feng XP etc. through the discriminating (Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics) of protein science prediction nasopharyngeal carcinoma radiotherapy reactive biomarker, cancer research (Cancer Res.), 2010, 70 (9): 3450-62).The nasopharyngeal carcinoma new cases that only Zhongshan Univ. Cancer Cure Center makes a definite diagnosis in In Guangzhou Area just reach 2400-2500 example/year.The knub position depths basis cranii of nasopharyngeal carcinoma, neck and masseter gap, very hidden, general surgical procedures cannot be adopted to excise focal zone lump.At present, radiotherapy as the first-selected treatment plan of nasopharyngeal carcinoma (see Ao Fan, Liao Yu reveals the correlation research [J] of .P53, Ki-67, Bcl-2, EGFR and radiosensitivity of nasopharyngeal carcinoma. and the practical combination of Chinese tradiational and Western medicine is clinical, 2010,10 (4): 4-5), especially good control action kou is had for the early stage nasopharyngeal carcinoma without distant metastasis, Synchronous chemoradiotherapy also becomes the standard treatment regimen (Ma Jun of local-advanced nasopharyngeal carcinoma gradually, the progress [J] for the treatment of of nasopharyngeal carcinoma. Zhongshan University's journal (medical science version), 2010,31 (2): 179-185).
Clinical treatment finds, even if Nasopharyngeal Carcinoma Patients belongs to by stages same, adopt same radiation therapy technology and identical radiotherapy dosage, still the patient of 20%-40% is had to occur recurring in launched field in 5 years, further investigation finds this mainly because the radiotherapy endogenous susceptibility of Different Individual tumour cell is different, show as radiation-sensitive or Radioresistence (see Ye Weijun, Min Huaqing, Cao Xinping, Deng the relation of the target biomolecules such as .P53 albumen, vascular endothelial growth factor and radiosensitivity of nasopharyngeal carcinoma. cancer, 2006,25 (9): 1168-1172).The main cause for the treatment of of nasopharyngeal carcinoma failure is caused to be distant metastasis and Radioresistence, simple radiotherapy is carried out to Nasopharyngeal Carcinoma Patients, its 5 years survival rates are about 50%, one of reason that treatment rate is not high i.e. Radioresistence is (see Xia Yunfei, Qian Jianyang, Zhang Enxi, practical nasopharyngeal carcinoma radiotherapy. medical publishing society of Peking University, Beijing, 2003,211-214).Therefore, excavate out the biomarker that specificity is good, highly sensitive, and develop quick, accurate, sensitive dependent diagnostic method, before Nasopharyngeal Carcinoma Patients receives treatment, its radiation sensitivity is evaluated, auxiliary enforcement nasopharyngeal carcinoma individualized treatment, becomes study hotspot, to raising nasopharyngeal carcinoma radiotherapy specific aim and validity, alleviate the unnecessary misery of patient and economical load simultaneously, significant.
At present for the diagnosis of nasopharyngeal carcinoma radiotherapy susceptibility, there is no diagnostic techniques or the unified evaluation index (gold standard) of authority.It is relevant to the radiation sensitivity of nasopharyngeal carcinoma that pertinent literature reports multiple protein mark.As PCNA(proliferating cell nuclear antigen) high expression level nasopharyngeal carcinoma radiotherapy susceptibility be proportionate, sensitivity during prediction nasopharyngeal carcinoma radiotherapy susceptibility is 94%, but specificity less than 32%(see the yellow East Sea, Tian Yongquan, Qiu Yuanzheng, Deng. application PCNA and survivin predicts the research [J] of nasopharyngeal carcinoma radiotherapy susceptibility. Tongji University's journal (medicine), 2006,, and there is the different report phenomenon inconsistent to the conclusion of different albumen candidate markers 27 (5): 39-42).The research of joint-detection in nasopharyngeal carcinoma radiotherapy susceptibility of protein marker was obtaining very large attention in recent years, find that 14-3-3 σ and maspin(mammary gland silk press down albumen as studied) downward, and the rise of GRP78 (78KDa glucose regulated protein) and Mn-SOD (manganese superoxide dismutase) to nasopharyngeal carcinoma Radioresistence significant correlation (see Feng XP etc., through the discriminating (Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics) of the reactive biomarker of protein science prediction nasopharyngeal carcinoma radiotherapy. cancer research (Cancer Res.) 2010,70 (9): 3450-62), there is good specificity and sensitivity, but the joint-detection of these four kinds of protein markers still need to rely on immunohistochemistry technique (see Cho WC etc. through serum protein group Epidemiological Analysis qualification serum amyloid protein as a kind of biomarker (Identification of serum amyloid a protein as a potentially useful biomarker to monitor relapse of nasopharyngeal cancer by serum proteomic profiling) of potential useful monitoring recurrent nasopharyngeal carcinoma. Clinical Cancer Research (Clin Cancer Res.) 2004,10 (1): 43-52), tissue of patient is taked to detect, testing cost is higher, cycle is longer, be difficult to the auxiliary diagnosis being widely used in clinical nasopharyngeal carcinoma radiotherapy susceptibility.
Abundant polypeptide moiety is rich in serum, be that variable body is for " diagnosis gold " for the serum polypeptide of " biologic garbage " by it is believed that in tradition, the bioinformation that serum polypeptide class mark discloses has caused the very big concern of researchist for the control of tumour.Based on serum polypeptide, and the clinical detection mode that combination is commonly used at present is early examined and individualized treatment auxiliary diagnosis tumour, obtains good Detection results.Villanueva etc. utilize serum polypeptide to have studied prostate cancer, mammary cancer and bladder cancer, have searched out the polypeptide fragment that tens can be used as tumor markers, have reached 100% to the predictablity rate of sample.
Patent US20090208921A1, discloses the tumor markers SSSYSKQFTSSTSYNRGDSTFESKSYKM for diagnosing prostate cancer, bladder cancer, but and this polypeptide unexposed can be used as a kind of mark judging nasopharyngeal carcinoma radiotherapy susceptibility.
Patent WO2009045552A1, discloses the method utilizing polypeptide marker SSSYSKQFTSSTSYNRGDSTFESKSYKM to carry out medical diagnosis on disease.Though this patent mentions this polypeptide marker, but be not specifically related to what disease and purposes, clearly do not embody relevant with nasopharyngeal carcinoma radiotherapy susceptibility.
Current serum polypeptide is mainly used in the analysis of prostate cancer, mammary cancer and bladder cancer etc., analyze the main immuno-mass spectrometry adopted based on antibody Human serum protein technology (immunomagnetic beads, antibody coupling solid phase carrier etc.), all there is testing cost costliness in these systems, operational requirement is higher, is unfavorable for the defect promoted.At present, still lack good, the highly sensitive biomarker of specificity, quick, accurate, sensitive dependent diagnostic method is carried out to nasopharyngeal carcinoma radiotherapy susceptibility.Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01 and ELISA kit thereof, for the radiation sensitivity auxiliary diagnosis in nasopharyngeal carcinoma individualized treatment.
Polypeptide marker SPG01 full length sequence provided by the invention is:
Ser-Ser-Ser-Tyr-Ser-Lys-Gln-Phe-Thr-Ser-Ser-Thr-Ser-Tyr-Asn-Ar g-Gly-Asp-Ser-Thr-Phe-Glu-Ser-Lys-Ser-Tyr-Lys-Met。
The present invention also provides the antibody that specific binding occurs with aforementioned polypeptides mark SPG01.Described antibody comprises monoclonal antibody and polyclonal antibody, and the method that described method for preparing monoclonal antibody can refer to patent CN102539767 is prepared.
Described polyclonal antibody is prepared in accordance with the following steps:
1) solid-phase synthesis is adopted to prepare synthetic peptide SPG01;
2) adopt carbodiimide coupling method that synthetic peptide SPG01 and the pure albumen of carrier proteins Bovine (BSA) coupling are prepared immunogen SPG01-BSA;
3) immunogen SPG01-BSA is carried out animal immune, adaptive immune serum, by purifying, obtain the anti-synthetic peptide SPG01 polyclonal antibody of concentration 3.8mg/mL, titre 1:100000.
Particularly, comprise the following steps: 1) adopt optimize carbodiimide coupling method synthetic peptide SPG01 is coupled to carrier proteins BSA(bovine serum albumin) on prepare conjugate SPG01-BSA, combined U V(UV scanning) and SDS-PAGE(polyacrylamide gel electrophoresis) monitor conjugate SPG01-BSA;
2) using the conjugate SPG01-BSA for preparing as immunity, in conjunction with Freund's complete adjuvant and Freund's incomplete adjuvant immunize New Zealand large ear rabbit, each conjugate antigen immune dosage is 1mg, and immunization ways is neck dorsal sc multi-point injection;
3) after 3 immunity, detect immunize rabbit venous blood titre, after reaching more than 1:10000, carotid artery is carried out to immunize rabbit and gets blood and collect immune whole blood, after centrifugation, collect rabbit immune serum;
4) the rabbit immune serum of Protein A affinity column antagonism synthetic peptide SPG01 is adopted to carry out purifying, prepare the polyclonal antibody of anti-synthetic peptide SPG01, detect the titre, specificity, susceptibility etc. of polyclonal antibody, obtain antiserum(antisera) polypeptide marker SPG01 antibody.
The present invention further provides the ELISA kit comprising above-mentioned polyclonal antibody.
Described ELISA kit comprise in coating buffer, 2%OVA, HRP ELIAS secondary antibody, TMB nitrite ion, 2M sulfuric acid and 96 hole elisa plates further one or more.
The present invention on the other hand, provides aforementioned polypeptides mark SPG01 and above-mentioned antibody for the preparation of the application detected in nasopharyngeal carcinoma radiotherapy susceptibility reagent.
Preferably, described is the ELISA kit comprising above-mentioned antibody for detecting nasopharyngeal carcinoma radiotherapy susceptibility reagent.
Described ELISA kit comprise in coating buffer, 2%OVA, HRP ELIAS secondary antibody, TMB nitrite ion, 2M sulfuric acid and 96 hole elisa plates further one or more.
Wherein, the antibody of described anti-synthetic peptide SPG01 is preferably the rabbit source polyclonal antibody of antiserum(antisera) polypeptide marker SPG01, and described HRP ELIAS secondary antibody is HRP-goat-anti rabbit.
The method that described ELISA kit detects polypeptide marker SPG01 comprises the steps:
1) collected specimens;
2) detect with described test kit;
3) analyzing and testing result.
Concrete steps are as follows: in 96 hole elisa plates, clinical detection serum and coating buffer bag spent the night, cleaning several times, close with 20%OVA solution, cleaning several times, add anti-synthetic peptide SPG01 polyclonal antibody, after optimal temperature is hatched, cleaning several times, add HRP ELIAS secondary antibody, cleaning several times, develop the color with TMB nitrite ion, stop with 2M sulfuric acid, 450nm detects OD value again.
In the present invention's preferred embodiment, described method comprises the steps:
1) in 96 orifice plates, add 95 μ L-80 μ L coating buffers, then add 5 μ L-20 μ L test serum samples, 4 DEG C of placements are spent the night; Use 100 μ L coating buffers as negative control simultaneously;
2) abandon coating buffer, clean 4-6 time with the PBST damping fluid 200-300 μ L of 0.01M, pH7.4, pat dry;
3) add the 2%OVA of 200-300 μ L, leave standstill in 37 DEG C and close 1.5-2 hour;
4) abandon confining liquid, clean 4-6 time with the PBST damping fluid 200-300 μ L of 0.01M, pH7.4, pat dry;
5) add resisting of the 80-120 μ anti-synthetic peptide SPG01 of L more, hatch 0.5-1 hour for 37 DEG C;
6) abandon antibody liquid, clean 4-6 time with the PBST damping fluid 200-300 μ L of 0.01M, pH7.4, pat dry;
7) add 80-120 μ L and resist many anti-ELIAS secondary antibody, hatch 0.5-1 hour for 37 DEG C;
8) abandon ELIAS secondary antibody liquid, clean 4-6 time with the PBST damping fluid 200-300 μ L of 0.01M, pH7.4, pat dry;
9) 80-120 μ LTMB nitrite ion is added, 37 DEG C of lucifuge colour developing 10-15min;
10) add 40-60 μ L2M sulfuric acid, termination reaction, in 5min, measure the absorbance (OD value) of wavelength 450nm with enzyme connection detector.
Beneficial effect of the present invention: (1) the present invention chooses nasopharyngeal carcinoma radiotherapy susceptibility associated serum polypeptide marker SPG01, prepare resisting of anti-synthetic peptide SPG01 more, and be applied to the analysis of 63 parts of serum in patients with nasopharyngeal samples (comprising radiotherapy sensitivity and radiotherapy resistance), analyze after testing and data statistics, find that the how anti-detection specific degree of this anti-synthetic peptide SPG01 reaches more than 90%, sensitivity, more than 70%, shows that this serum polypeptide mark obviously can be distinguished nasopharyngeal carcinoma radiotherapy sensitivity and resist with radiotherapy.There is provided thus containing this how anti-ELISA kit and detection method.
(2) carbodlimide method of the present invention has carried out the optimization of coupling time, ingredient proportion, Loading sequence to carbodiimide polypeptide immune antigen coupling method emphasis, and combines UV and calculate coupling ratio, SDS-PAGE and calculates Conjugate ratio, and antithesis connection is really monitored.Carry out method validation with the improvement on synthesis sample of different molecular weight size, amino acid composition and end amino acid, all obtained specific antibody, illustrated that the inventive method suitability is wide, reduce the risk of animal immune failure.The present invention propose with UV coupling ratio and SDS-PAGE Conjugate ratio association evaluation coupled product, for instruct animal immune test there is good practical value.
(3) many anti-high specificities for polypeptide marker SPG01 provided by the present invention; The detection method simple operating steps of polypeptide marker SPG01 is controlled, is beneficial to clinical detection; The enzyme connection detector detection of high-throughput, low cost can be carried out.
(4) ELISA kit that the present invention is based on this serum polypeptide mark SPG01 can in enforcement nasopharyngeal carcinoma individualized treatment system, radiation sensitivity evaluation is carried out before radiotherapy is received to Nasopharyngeal Carcinoma Patients, play the effect of auxiliary diagnosis, there is obvious practicality.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of the synthetic peptide SPG01 standard model of serum polypeptide mark of the present invention;
Fig. 2 A is SPG01-BSAD UV scanning monitoring result, and B is the SDS-PAGE electrophoresis monitoring result of SPG01-BSAD, and wherein, BSA is bovine serum albumin; SPG01-BSA is the conjugate of SPG01 and BSA;
Fig. 3 is the ROC curve of detection nasopharyngeal carcinoma radiotherapy of the present invention sensitivity group and radiotherapy resistance group;
Fig. 4 A is the box figure of detection nasopharyngeal carcinoma radiotherapy of the present invention sensitivity group; B is the box figure of radiotherapy resistance group.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.SPG01 synthetic peptide entrusts Shanghai Qiangyao Biotechnology Co., Ltd. to synthesize.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
Embodiment 1 carbodiimide coupling method prepares SPG01-BSA conjugate
1) SPG01, BSA, EDC(carbodiimide is balanced) to room temperature, use 0.1mol/L respectively, the MES biological buffer of pH6.0 is mixed with 5mg/mL, 10mg/mL, 10mg/mL; SPG01 full length sequence is:
Ser-Ser-Ser-Tyr-Ser-Lys-Gln-Phe-Thr-Ser-Ser-Thr-Ser-Tyr-As n-Arg-Gly-Asp-Ser-Thr-Phe-Glu-Ser-Lys-Ser-Tyr-Lys-Met。The mass spectrum of the synthetic peptide SPG01 standard model of serum polypeptide mark as shown in Figure 1.
2) SPG01 and BSA is respectively drawn 1mg to be pre-mixed in 5mL phial, then carry out coupling in the 5mL phial being added to 1mg EDC, react and carry out in 25 DEG C of thermostat containers, coupling 12 hours under moderate-speed mixer;
3), after reaction terminates, conjugate is placed in the dialysis tubing of 12-14KDa, dialyses 24 hours with the PBS of 0.02mol/L, pH7.4;
4) the SPG01-BSA conjugate after dialysis is collected, combined U V(UV scanning) and SDS-PAGE(polyacrylamide gel electrophoresis) monitor conjugate SPG01-BSA.
5) UV and SDS-PAGE monitoring result as shown in Figure 2: UV result shows: the UV scanning curve of polypeptide SPG01 and BSA is obviously different, the characteristic absorption wavelength of BSA is at 280nm place, the characteristic absorbance of SPG01 is at 278nm place, the UV curve of coupled antigen SPG01-BSA moves on the whole than BSA, and in the rising more obvious than BSA of the peak valley at 250nm place, illustrate that linked reaction changes the UV characteristic of BSA, demonstrate polypeptide success coupling (UV figure).SDS-PAGE result shows, and conjugate band presents diffuse type (empty collimation mark knowledge), is significantly different from the band (SDS-PAGE) of BSA.Ultraviolet and electrophoresis monitoring result show that polypeptide SPG01 is successfully coupled on BSA.
The preparation method of the anti-synthetic peptide SPG01 polyclonal antibody of embodiment 2
1) adopt conjugate SPG01-BSA as immunogen, in conjunction with Freund's complete adjuvant and Freund's incomplete adjuvant, immunize New Zealand large ear rabbit;
2) after the 3rd immunity 3 days, detect immunize rabbit venous blood titre, titre reaches at more than 1:10000 got blood standard;
3) carry out carotid artery to immunize rabbit to get blood and collect immune whole blood, after centrifugation, collect rabbit immune serum;
4) the rabbit immune serum of Protein A affinity column antagonism synthetic peptide SPG01 is adopted to carry out purifying, prepare the polyclonal antibody of anti-synthetic peptide SPG01, detect the concentration 3.8mg/mL of this polyclonal antibody, titre 1:100000, obtain antiserum(antisera) polypeptide marker SPG01 antibody.Explanation immune effect is better, containing the specific antibody for anti-synthetic peptide SPG01 in immunize rabbit serum.
The assembling of the ELSIA test kit of embodiment 3 polypeptide marker SPG01
1) coating buffer: the carbonate buffer solution of 0.1M pH9.6;
2) 2%OVA:2g/100mL ovalbumin;
3) HRP ELIAS secondary antibody: be century biotechnology company purchased from Beijing health
4) TMB nitrite ion: A liquid hydrogen peroxide, B liquid TMB(tetramethyl benzidine);
5) 2M sulfuric acid;
6) 96 hole elisa plates.
The ELISA detection method of embodiment 4 polypeptide marker SPG01
Sample: the responsive serum 33 parts of the clinical nasopharyngeal carcinoma radiotherapy made a definite diagnosis after radiotherapy, nasopharyngeal carcinoma radiotherapy resists serum 30 parts.
Testing process:
1) in 96 orifice plates, add 90 μ L coating buffers, then add 10 μ L test serum samples, 4 DEG C of placements are spent the night; Use 100 μ L coating buffers as negative control simultaneously;
2) abandon coating buffer, clean 5 times with the PBST damping fluid 250 μ L of 0.01M, pH7.4, pat dry;
3) add the 2%OVA of 200-300 μ L, leave standstill in 37 DEG C and close 2 hours;
4) abandon confining liquid, clean 5 times with the PBST damping fluid 250 μ L of 0.01M, pH7.4, pat dry;
5) add resisting of the 100 μ anti-synthetic peptide SPG01 of L more, hatch 1 hour for 37 DEG C;
6) abandon antibody liquid, clean 5 times with the PBST damping fluid 250 μ L of 0.01M, pH7.4, pat dry;
7) add 100 μ L and resist many anti-ELIAS secondary antibody, hatch 40min for 37 DEG C;
8) abandon ELIAS secondary antibody liquid, clean 5 times with the PBST damping fluid 250 μ L of 0.01M, pH7.4, pat dry;
9) 100 μ LTMB nitrite ions (each 50 μ L of A, B liquid) are added, 37 DEG C of lucifuge colour developing 15min;
10) add 50 μ L2M sulfuric acid, termination reaction, in 5min, measure the absorbance (OD value) of wavelength 450nm with enzyme connection detector.
Result is through statistics, and as the ROC curve display of Fig. 3, the susceptibility of this test kit reaches 73.3%, and specific degree reaches 93.9%; T check analysis P=1.1319E-05<0.05, illustrates that polypeptide marker SPG01 obviously can distinguish two groups of samples; Box figure shows (Fig. 4), and two groups of samples are significantly separated, and its median difference significantly, is intersected less between two groups.Above analytic explanation serum polypeptide mark of the present invention SPG01 and ELISA detection kit significantly can distinguish nasopharyngeal carcinoma radiotherapy sensitivity and radiotherapy resistance group, and high specificity is reproducible.
Compared with patent US20090208921A1, the present invention is as a kind of mark judging nasopharyngeal carcinoma radiotherapy susceptibility using polypeptide SPG01, for disease and the bladder cancer described in US20090208921A1, prostate cancer different, and the present invention is more specifically to and radiotherapy responsive to the radiotherapy of nasopharyngeal carcinoma resists and distinguish, respond well, and do not have relevant report in that patent.
Compared with patent WO2009045552A1, though WO2009045552A1 mentions this mark, be not specifically related to what disease and what purposes, clearly do not embody relevant with nasopharyngeal carcinoma radiotherapy susceptibility.The invention provides with SPG01 is the ELISA kit of nasopharyngeal carcinoma radiotherapy sensitivity label thing, in purposes, very concrete in using method, these groping all through great many of experiments condition, and does not have relevant report in that patent.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (4)

1. nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01 is for the preparation of the application detected in nasopharyngeal carcinoma radiotherapy susceptibility reagent, and described polypeptide marker SPG01 full length amino acid sequence is as shown in SEQ ID No.1.
2. with nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01 be antigen, with the antibody of this polypeptide generation specific binding for the preparation of the application detected in nasopharyngeal carcinoma radiotherapy susceptibility reagent, described polypeptide marker SPG01 full length amino acid sequence is as shown in SEQ ID No.1.
3. application according to claim 2, is characterized in that, described antibody is polyclonal antibody, and it is prepared in accordance with the following steps:
1) solid-phase synthesis is adopted to prepare synthetic peptide SPG01;
2) adopt carbodiimide coupling method that synthetic peptide SPG01 and the pure albumen coupling of carrier proteins Bovine are prepared immunogen SPG01-BSA;
3) immunogen SPG01-BSA is carried out animal immune, adaptive immune serum, by purifying, obtain the anti-synthetic peptide SPG01 polyclonal antibody of concentration 3.8mg/mL, titre 1:100000.
4. application according to claim 3, is characterized in that, step 2) in synthetic peptide SPG01, bovine serum albumin, carbodiimide mass ratio be 1:1:1, coupling time is 10-12h.
CN201310210634.5A 2013-05-30 2013-05-30 Radiosensitive polypeptide mark SPG01 for nasopharynx cancer and ELISA (Enzyme-Linked Immuno Sorbent Assay) kit thereof Active CN103304658B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009045552A1 (en) * 2007-10-05 2009-04-09 Becton, Dickinson And Company System and method for diagnosing diseases
US20090208921A1 (en) * 2005-08-16 2009-08-20 Sloan Kettering Institute For Cancer Research Methods of detection of cancer using peptide profiles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090208921A1 (en) * 2005-08-16 2009-08-20 Sloan Kettering Institute For Cancer Research Methods of detection of cancer using peptide profiles
WO2009045552A1 (en) * 2007-10-05 2009-04-09 Becton, Dickinson And Company System and method for diagnosing diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Identifying FGA Peptides as Nasopharyngeal Carcinoma-Associated Biomarkers by Magnetic Beads;Ya-Lan Tao;《Journal of Cellular Biochemistry》;20121231;第113卷;表5,摘要 *

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