CN103304658A - 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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CN103304658A
CN103304658A CN2013102106345A CN201310210634A CN103304658A CN 103304658 A CN103304658 A CN 103304658A CN 2013102106345 A CN2013102106345 A CN 2013102106345A CN 201310210634 A CN201310210634 A CN 201310210634A CN 103304658 A CN103304658 A CN 103304658A
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spg01
antibody
polypeptide
nasopharyngeal carcinoma
synthetic peptide
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CN103304658B (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 test kit thereof
Technical field
The present invention relates to the biotechnology detection field, specifically, relate to a kind of nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01 and ELISA test 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 called again " canton tumor (Canton tumor) ".Although worldwide nasopharyngeal carcinoma belongs to rare tumor, sickness rate or mortality ratio in most countries all are lower than 1/100000, but at the nasopharyngeal carcinoma annual morbidity of South China and south east asia up to 15~50/100000, and histopathology often be diagnosed as II phase of WHO regulation or III phase (referring to 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).Only the nasopharyngeal carcinoma new cases made a definite diagnosis at In Guangzhou Area of Zhongshan Univ. Cancer Cure Center just nearly the 2400-2500 example/year.Knub position depths basis cranii, neck and the masseter gap of nasopharyngeal carcinoma, very hidden, can't adopt the conventional surgical operation that the focal zone lump is excised.At present, radiotherapy as the first-selected treatment plan of nasopharyngeal carcinoma (referring to 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 for the early stage nasopharyngeal carcinoma without distant metastasis good control action kou is arranged, the Synchronous Radio chemotherapy also becomes the standard care mode (Ma Jun of local-advanced nasopharyngeal carcinoma gradually, the progress for the treatment of of nasopharyngeal carcinoma [J]. Zhongshan University's journal (medical science version), 2010,31 (2): 179-185).
Clinical treatment is found, even it is by stages same that Nasopharyngeal Carcinoma Patients belongs to, adopt same radiation therapy technology and identical radiotherapy dosage, still there is the patient of 20%-40% in 5 years, to occur recurring in the launched field, further investigation finds that this mainly is because the radiotherapy endogenous susceptibility of Different Individual tumour cell is different, show as radiation-sensitive or Radioresistence (referring to Ye Weijun, Min Huaqing, Cao Xinping, in .P53 albumen, the relation of the target biomolecules such as vascular endothelial growth factor and radiosensitivity of nasopharyngeal carcinoma. cancer, 2006,25 (9): 1168-1172).The main cause that causes the treatment of nasopharyngeal carcinoma failure is distant metastasis and Radioresistence, Nasopharyngeal Carcinoma Patients is carried out simple radiotherapy, its 5 years survival rates are about 50%, one of reason that treatment rate is not high be Radioresistence (referring to Xia Yunfei, Qian Jianyang, Zhang Enxi, practical nasopharyngeal carcinoma radiotherapy is learned. medical publishing society of Peking University, Beijing, 2003,211-214).Therefore, excavate out good, the highly sensitive biomarker of specificity, and develop quick, accurate, sensitive dependent diagnostic method, before Nasopharyngeal Carcinoma Patients receives treatment, its radiation sensitivity is estimated, the auxiliary nasopharyngeal carcinoma individualized treatment of implementing has become study hotspot, to improving nasopharyngeal carcinoma radiotherapy specific aim and validity, alleviate simultaneously the unnecessary misery of patient and economical load, significant.
For the diagnosis of nasopharyngeal carcinoma radiotherapy susceptibility, there is no authority's diagnostic techniques or unified evaluation index (gold standard) at present.Pertinent literature has reported that the multiple protein mark is relevant with the radiation sensitivity of nasopharyngeal carcinoma.Such as the PCNA(proliferating cell nuclear antigen) high expression level nasopharyngeal carcinoma radiotherapy susceptibility be proportionate, sensitivity during prediction nasopharyngeal carcinoma radiotherapy susceptibility is 94%, but the not enough 32%(of specificity is referring to the yellow East Sea, Tian Yongquan, Qiu Yuanzheng, etc. use the research [J] of PCNA and survivin prediction nasopharyngeal carcinoma radiotherapy susceptibility. Tongji University's journal (medicine), 2006,27 (5): 39-42), and exist different reports to the inconsistent phenomenon of the conclusion of different albumen candidate markers.The research of the joint-detection of protein marker in nasopharyngeal carcinoma radiotherapy susceptibility obtains very large attention in recent years, as study and find that 14-3-3 σ and maspin(mammary gland silk press down albumen) downward modulation, and the rise of GRP78 (78KDa glucose regulated protein) and Mn-SOD (manganese superoxide dismutase) to nasopharyngeal carcinoma Radioresistence significant correlation (referring to Feng XP etc., discriminating (Identification of biomarkers for predicting nasopharyngeal carcinoma response to radiotherapy by proteomics) through the reactive biomarker of protein science prediction nasopharyngeal carcinoma radiotherapy. cancer research (Cancer Res.) 2010,70 (9): 3450-62), has good specificity and sensitivity, but the joint-detection of these four kinds of protein markers still need to rely on immunohistochemistry technique (referring to Cho WC etc. identify that through serum protein group Epidemiological Analysis serum amyloid protein is 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), take tissue of patient to detect, testing cost is higher, cycle is longer, is difficult to be widely used in the auxiliary diagnosis of clinical nasopharyngeal carcinoma radiotherapy susceptibility.
Be rich in abundant polypeptide composition in the serum, be it is believed that in the tradition be the serum polypeptide of " biologic garbage " variable body is for " diagnosis gold ", the bioinformation that serum polypeptide class mark discloses has caused researchist's very big concern for the control of tumour.Based on serum polypeptide, and in conjunction with clinical detection mode at present commonly used tumour is early examined and the individualized treatment auxiliary diagnosis, obtained good detection effect.Villanueva etc. utilize serum polypeptide to study prostate cancer, mammary cancer and bladder cancer, have searched out tens polypeptide fragments that can be used as tumor markers, and the predictablity rate of sample is reached 100%.
Patent US20090208921A1 disclose the tumor markers SSSYSKQFTSSTSYNRGDSTFESKSYKM that is used for diagnosing prostate cancer, bladder cancer, but also unexposed this polypeptide can be used as a kind of mark of judging nasopharyngeal carcinoma radiotherapy susceptibility.
Patent WO2009045552A1 discloses the method for utilizing polypeptide marker SSSYSKQFTSSTSYNRGDSTFESKSYKM to carry out medical diagnosis on disease.Though this patent is mentioned this polypeptide marker, but be not specifically related to what disease and purposes, more clearly do not embody relevant with nasopharyngeal carcinoma radiotherapy susceptibility.
Present serum polypeptide is mainly used in the analysis of prostate cancer, mammary cancer and bladder cancer etc., analyze the main immune substance spectrometry that adopts based on the affine isolation technique of antibody (immunomagnetic beads, antibody coupling solid phase carrier etc.), these systems all exist testing cost expensive, operational requirement is higher, is unfavorable for the defective of promoting.At present, still lack good, the highly sensitive biomarker of specificity, nasopharyngeal carcinoma radiotherapy susceptibility is carried out quick, accurate, sensitive dependent diagnostic method.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 test kit thereof, be used for the radiation sensitivity auxiliary diagnosis of 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 described method for preparing monoclonal antibody can be prepared with reference to the method for patent CN102539767.
Described polyclonal antibody prepares in accordance with the following steps:
1) adopt solid-phase synthesis to prepare synthetic peptide SPG01;
2) adopt the carbodiimide coupling method that synthetic peptide SPG01 and carrier proteins bovine serum albumin (BSA) coupling are prepared immunogen SPG01-BSA;
3) immunogen SPG01-BSA is carried out animal immune, adaptive immune serum by purifying, obtains the anti-synthetic peptide SPG01 polyclonal antibody of concentration 3.8mg/mL, titre 1:100000.
Particularly, may further comprise the steps: 1) adopt the carbodiimide coupling method of optimizing that synthetic peptide SPG01 is coupled to carrier proteins BSA(bovine serum albumin) upper preparation conjugate SPG01-BSA, combined U V(UV scanning) and the SDS-PAGE(polyacrylamide gel electrophoresis) monitoring conjugate SPG01-BSA;
2) with the conjugate SPG01-BSA for preparing as immunity, in conjunction with Freund's complete adjuvant and Freunds incomplete adjuvant immunity New Zealand large ear rabbit, each conjugate antigen immune dosage is 1mg, immunization ways is the subcutaneous multi-point injection of nape section;
3) after 3 immunity, detect immunize rabbit venous blood titre, reach 1:10000 above after, immunize rabbit is carried out carotid artery gets blood and collect immune whole blood, collect the rabbit immune serum after the centrifugation;
4) adopt the rabbit immune serum of Protein A affinity column antagonism synthetic peptide SPG01 to carry out purifying, the polyclonal antibody for preparing anti-synthetic peptide SPG01, the titre of detection polyclonal antibody, specificity, susceptibility etc. obtain antiserum(antisera) polypeptide marker SPG01 antibody.
The present invention further provides the ELISA test kit that comprises above-mentioned polyclonal antibody.
Described ELISA test kit further comprises one or more in coating buffer, 2%OVA, HRP ELIAS secondary antibody, TMB nitrite ion, 2M sulfuric acid and the 96 hole elisa plates.
The present invention provides aforementioned polypeptides mark SPG01 and above-mentioned antibody for the preparation of the application that detects in the nasopharyngeal carcinoma radiotherapy susceptibility reagent on the other hand.
Preferably, described is the ELISA test kit that comprises above-mentioned antibody for detection of nasopharyngeal carcinoma radiotherapy susceptibility reagent.
Described ELISA test kit further comprises one or more in coating buffer, 2%OVA, HRP ELIAS secondary antibody, TMB nitrite ion, 2M sulfuric acid and the 96 hole elisa plates.
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 test 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, spend the night clinical detection serum and coating buffer are coated, clean several times, seal with 20%OVA solution, clean several times, add anti-synthetic peptide SPG01 polyclonal antibody, after optimal temperature is hatched, clean several times, add the HRP ELIAS secondary antibody, clean several times, with the colour developing of TMB nitrite ion, stop with 2M sulfuric acid, 450nm detects the OD value again.
In preferred embodiment of the present invention, described method comprises the steps:
1) in 96 orifice plates, add 95 μ L-80 μ L coating buffers, add again 5 μ L-20 μ L test serum samples, 4 ℃ of placements are spent the night; Use simultaneously 100 μ L coating buffers as negative control;
2) abandon coating buffer, use the PBST damping fluid 200-300 μ L of 0.01M, pH7.4 to clean 4-6 time, pat dry;
3) add the 2%OVA of 200-300 μ L, leave standstill sealing 1.5-2 hour in 37 ℃;
4) abandon confining liquid, use the PBST damping fluid 200-300 μ L of 0.01M, pH7.4 to clean 4-6 time, pat dry;
5) add the how anti-of the anti-synthetic peptide SPG01 of 80-120 μ L, hatched 0.5-1 hour for 37 ℃;
6) abandon antibody liquid, use the PBST damping fluid 200-300 μ L of 0.01M, pH7.4 to clean 4-6 time, pat dry;
7) add the anti-many anti-ELIAS secondary antibody of 80-120 μ L, hatched 0.5-1 hour for 37 ℃;
8) abandon ELIAS secondary antibody liquid, use the PBST damping fluid 200-300 μ L of 0.01M, pH7.4 to clean 4-6 time, pat dry;
9) add 80-120 μ LTMB nitrite ion, 37 ℃ of lucifuge colour developing 10-15min;
10) add 40-60 μ L2M sulfuric acid, termination reaction joins the absorbance (OD value) that detector is measured wavelength 450nm with enzyme in the 5min.
Beneficial effect of the present invention: (1) the present invention chooses the relevant serum polypeptide mark SPG01 of nasopharyngeal carcinoma radiotherapy susceptibility, prepare the how anti-of anti-synthetic peptide SPG01, and be applied to the analysis of 63 parts of serum in patients with nasopharyngeal samples (comprise radiotherapy responsive and radiotherapy resistance), analyze after testing and data statistics, find how anti-detection specific degree reaches more than 90% this anti-synthetic peptide SPG01, sensitivity shows that more than 70% this serum polypeptide mark can obviously be distinguished the nasopharyngeal carcinoma radiotherapy sensitivity and radiotherapy is resisted.Provide thus and contain this many anti-ELISA test kit and detection methods.
(2) carbodlimide method of the present invention has carried out coupling time, ingredient proportion, application of sample optimization sequentially to carbodiimide polypeptide immune antigen coupling method emphasis, and associating UV calculates coupling ratio, SDS-PAGE calculates the coupling rate, and antithesis connects really monitors.Synthetic polypeptide sample with different molecular weight size, amino acid composition and end amino acid has carried out method validation, all obtains specific antibody, illustrates that the inventive method suitability is wide, reduces the risk of animal immune failure.The present invention propose with UV coupling ratio and SDS-PAGE coupling rate association evaluation coupled product, for instructing the animal immune experiment to have 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; Can carry out high-throughput, cheaply enzyme connection detector detection.
(4) the present invention is based on the ELISA test kit of this serum polypeptide mark SPG01 can be in implementing nasopharyngeal carcinoma individualized treatment system, to carrying out the radiation sensitivity evaluation before the Nasopharyngeal Carcinoma Patients reception radiotherapy, play the effect of auxiliary diagnosis, have obvious practicality.
Description of drawings
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 the responsive group of detection nasopharyngeal carcinoma radiotherapy of the present invention and radiotherapy resistance group;
Fig. 4 A is the box figure of the responsive group of detection nasopharyngeal carcinoma radiotherapy of the present invention; B is the box figure of radiotherapy resistance group.
Embodiment
Following examples are used for explanation 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, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.The SPG01 synthetic peptide entrusts the Shanghai bio tech ltd of shining by force synthetic.
If do not specialize, the conventional means that used technique means is well known to those skilled in the art among the embodiment.
Embodiment 1 carbodiimide coupling method prepares the SPG01-BSA conjugate
1) balance SPG01, BSA, EDC(carbodiimide) to room temperature, use respectively 0.1mol/L, the MES biological buffer of pH6.0 is mixed with 5mg/mL, 10mg/mL, 10mg/mL; The 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 are respectively drawn 1mg and be pre-mixed in the 5mL phial, be added in the 5mL phial of 1mg EDC again and carry out coupling, reaction is carried out in 25 ℃ of thermostat containers, and middling speed stirs lower coupling 12 hours;
3) after reaction finishes, conjugate is placed the dialysis tubing of 12-14KDa, use 0.02mol/L, the PBS dialysis of pH7.4 24 hours;
4) the SPG01-BSA conjugate after the collection dialysis, combined U V(UV scanning) and the SDS-PAGE(polyacrylamide gel electrophoresis) monitoring conjugate SPG01-BSA.
5) UV and SDS-PAGE monitoring result are as shown in Figure 2: UV result shows: polypeptide SPG01 is obviously different from the UV scanning curve of BSA, the characteristic absorption wavelength of BSA is at the 280nm place, the characteristic absorbance of SPG01 is at the 278nm place, the UV curve of coupled antigen SPG01-BSA moves on the whole than BSA, and at the peak valley at 250nm place than the obvious rising of BSA, illustrate that linked reaction has changed the UV characteristic of BSA, verified polypeptide success coupling (UV figure).SDS-PAGE result's demonstration, the conjugate band presents diffuse type (empty collimation mark knowledge), significantly is different from the band (SDS-PAGE) of BSA.Ultraviolet and electrophoresis monitoring result show that polypeptide SPG01 successfully is coupled on the BSA.
The preparation method of embodiment 2 anti-synthetic peptide SPG01 polyclonal antibodies
1) adopt conjugate SPG01-BSA as immunogen, in conjunction with Freund's complete adjuvant and Freunds incomplete adjuvant, immune New Zealand large ear rabbit;
2) after the 3rd immunity 3 days, detect immunize rabbit venous blood titre, titre reaches the blood standard of getting more than 1:10000;
3) immunize rabbit is carried out carotid artery and get blood and collect immune whole blood, collect the rabbit immune serum after the centrifugation;
4) adopt the rabbit immune serum of Protein A affinity column antagonism synthetic peptide SPG01 to carry out purifying, the polyclonal antibody for preparing anti-synthetic peptide SPG01, detect concentration 3.8mg/mL, the titre 1:100000 of this polyclonal antibody, obtain antiserum(antisera) polypeptide marker SPG01 antibody.Illustrate that immune effect is better, contain the specific antibody for anti-synthetic peptide SPG01 in the 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 available 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: 33 parts of the responsive serum of the clinical nasopharyngeal carcinoma radiotherapy of making a definite diagnosis behind radiotherapy, nasopharyngeal carcinoma radiotherapy is resisted 30 parts of serum.
Testing process:
1) in 96 orifice plates, add 90 μ L coating buffers, add again 10 μ L test serum samples, 4 ℃ of placements are spent the night; Use simultaneously 100 μ L coating buffers as negative control;
2) abandon coating buffer, use the PBST damping fluid 250 μ L of 0.01M, pH7.4 to clean 5 times, pat dry;
3) add the 2%OVA of 200-300 μ L, leave standstill sealing 2 hours in 37 ℃;
4) abandon confining liquid, use the PBST damping fluid 250 μ L of 0.01M, pH7.4 to clean 5 times, pat dry;
5) add the how anti-of the anti-synthetic peptide SPG01 of 100 μ L, hatched 1 hour for 37 ℃;
6) abandon antibody liquid, use the PBST damping fluid 250 μ L of 0.01M, pH7.4 to clean 5 times, pat dry;
7) add the anti-many anti-ELIAS secondary antibody of 100 μ L, hatch 40min for 37 ℃;
8) abandon ELIAS secondary antibody liquid, use the PBST damping fluid 250 μ L of 0.01M, pH7.4 to clean 5 times, pat dry;
9) add 100 μ LTMB nitrite ions (A, each 50 μ L of B liquid), 37 ℃ of lucifuge colour developing 15min;
10) add 50 μ L2M sulfuric acid, termination reaction joins the absorbance (OD value) that detector is measured wavelength 450nm with enzyme in the 5min.
The result by statistics, such as the ROC curve display of Fig. 3, the susceptibility of this test kit reaches 73.3%, specific degree reaches 93.9%; T check analysis P=1.1319E-05<0.05 illustrates that polypeptide marker SPG01 can obviously distinguish two groups of samples; Box figure shows (Fig. 4), and two groups of samples are significantly separated, and its median differs significantly, intersects less between two groups.Above analytic explanation serum polypeptide mark of the present invention SPG01 and ELISA detection kit can significantly be distinguished nasopharyngeal carcinoma radiotherapy sensitivity and radiotherapy resistance group, high specificity, good reproducibility.
US20090208921A1 compares with patent, the present invention is as a kind of mark of judging nasopharyngeal carcinoma radiotherapy susceptibility with polypeptide SPG01, for disease and the described bladder cancer of US20090208921A1, prostate cancer different, and the present invention is more specifically to responsive the resistance with radiotherapy of the radiotherapy of nasopharyngeal carcinoma is distinguished, respond well, and in this patent, relevant report is not arranged.
WO2009045552A1 compares with patent, though WO2009045552A1 mentions this mark, is not specifically related to what disease and what purposes, does not more clearly embody relevant with nasopharyngeal carcinoma radiotherapy susceptibility.The invention provides the ELISA test kit as nasopharyngeal carcinoma radiotherapy sensitivity label thing take SPG01, in purposes, very concrete on the using method, these all pass through groping of great many of experiments condition, and in this patent relevant report are not arranged.
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 all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Figure IDA00003276024900011

Claims (10)

1. nasopharyngeal carcinoma radiotherapy susceptibility polypeptide marker SPG01, its full length amino acid sequence is shown in SEQ ID No.1.
One kind take the described polypeptide marker SPG01 of claim 1 as antigen, with the antibody of this polypeptide generation specific binding.
3. antibody according to claim 2 is characterized in that, described antibody is polyclonal antibody, and it prepares in accordance with the following steps:
1) adopt solid-phase synthesis to prepare synthetic peptide SPG01;
2) adopt the carbodiimide coupling method that synthetic peptide SPG01 and the coupling of carrier proteins bovine serum albumin are prepared immunogen SPG01-BSA;
3) immunogen SPG01-BSA is carried out animal immune, adaptive immune serum by purifying, obtains the anti-synthetic peptide SPG01 polyclonal antibody of concentration 3.8mg/mL, titre 1:100000.
4. antibody according to claim 3 is characterized in that step 2) in the mass ratio of synthetic peptide SPG01, bovine serum albumin, carbodiimide be that 1:1:1, coupling time are 10-12h.
5. an ELISA test kit is characterized in that, comprises described antibody of claim 2-4.
6. ELISA test kit according to claim 5 is characterized in that, further comprises in coating buffer, 2%OVA, HRP ELIAS secondary antibody, TMB nitrite ion, 2M sulfuric acid and the 96 hole elisa plates one or more.
7. each described antibody of polypeptide marker SPG01 claimed in claim 1, claim 2-4 is for the preparation of the application that detects in the nasopharyngeal carcinoma radiotherapy susceptibility reagent.
8. application according to claim 7 is characterized in that, utilizes each described antibody of claim 2-4 to prepare the ELISA test kit.
9. application according to claim 8 is characterized in that, described ELISA test kit further comprises one or more in coating buffer, 2%OVA, HRP ELIAS secondary antibody, TMB nitrite ion, 2M sulfuric acid and the 96 hole elisa plates.
10. according to claim 9 application is characterized in that described antibody is rabbit source polyclonal antibody, and described HRP ELIAS secondary antibody is HRP-goat-anti rabbit.
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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
YA-LAN TAO: "Identifying FGA Peptides as Nasopharyngeal Carcinoma-Associated Biomarkers by Magnetic Beads", 《JOURNAL OF CELLULAR BIOCHEMISTRY》 *

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