CN102520174A - Method related to detection of lung cancer by using lung cancer diagnostic reagent kit - Google Patents
Method related to detection of lung cancer by using lung cancer diagnostic reagent kit Download PDFInfo
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- CN102520174A CN102520174A CN2011103282947A CN201110328294A CN102520174A CN 102520174 A CN102520174 A CN 102520174A CN 2011103282947 A CN2011103282947 A CN 2011103282947A CN 201110328294 A CN201110328294 A CN 201110328294A CN 102520174 A CN102520174 A CN 102520174A
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Abstract
The invention relates to the field of biotechnology and medicine, in particular to a method related to the detection of a lung cancer by using a lung cancer diagnostic reagent kit, wherein a protein S100A2 is produced through purification by using a gene recombination technology, the purified protein S100A2 is injected to the inside of an animal body so as to induce and produce a polyclonal antibody specifically combined with the protein S100A2. In addition, the invention provides the lung cancer diagnostic reagent kit, which comprises a container, a label and a specific antibody resist to the protein S100A2, wherein the container contains the protein S100A2, and the label is used for indicating that the reagent kit is used for lung cancer diagnosis. The reagent kit alternatively contains immunohistochemistry related reagents, such as a PBS (Phosphate Buffered Solution), or a 0.01mol/L sodium citrate buffered solution, or a 3% H2O2 solution or a horseradish peroxidase detection reagent. The invention makes advanced researches and large-scale verification on the relevancy between S100A2 and the lung cancer, a detection reagent capable of being applied clinically is worked out, the lung cancer can be diagnosed, and an individualized adjuvant therapy can be provided to a patient.
Description
[technical field]
The present invention relates to biotechnology and medical domain, specifically is the detection method that a kind of relevant pulmonary cancer diagnosis kit is used for lung cancer.
[background technology]
Lung cancer is one of present modal malignant tumour, also is the fastest tumour of global M & M increase.In the world, lung cancer ranks first in male sex's kinds of tumor, in women's kinds of tumor, occupy two, three.Probably have every year 1200000 patients to be diagnosed as lung cancer, 1,100,000 people die from lung cancer.Lung cancer can be divided into two big types according to its histopathology: ED-SCLC and non-small cell lung cancer.Non-small cell lung cancer comprises squamous cell carcinoma, gland cancer, large cell carcinoma etc. again.Up to now, the screening lung cancer scheme in the whole world is still immature.Therefore, early screening is own through becoming the great problem in science that lung cancer control urgent need solves with diagnosis.The discovery of tumor markers and rational Application are the prerequisites of tumour early detection and individualized treatment thereof.
At present the pathogenesis of relevant lung cancer it be unclear that, and research shows directly to start with from the protein integral level to be studied lung cancer and might seek out the more suitably molecular marker of pulmonary cancer diagnosis, for the diagnosis prevention and the treatment of lung cancer provides foundation.Calbindin is the acid albumen of low-molecular-weight, and 17 family members are arranged.Wherein S100 family has 13.In the S100 protein family, relevant with tumour have S100A2, S100A4, S100A6 and a S100B.Original research thinks that S100A2 is that unique and tumour generate the albumen that is negative correlation in the S100 protein family, because its expression in some tumor tissues and cell is low or lack, so be called as tumor suppressor gene.Detect S100A2 in cervical carcinoma, cancer of pancreas, the cancer of the esophagus and the lung cancer and express and to increase and also be reported in recently, and relevant with the progress and the prognosis of tumour.
In a word, known S100A2 expresses variant in some tumour, and is also different aspect its effect such as generation, development and transfer in different tumours, and still has some disputes at present.Therefore, the correlativity that further investigation S100A2 and lung cancer take place, develop and shift, and it is significant to disclose its clinical value.The present invention develops the detectable that can supply clinical practice on the early-stage Study basis, with the correlativity of research in depth with extensive checking S100A2 and lung cancer, cancer is diagnosed and better auxiliary treating method is provided for individual patient.
[summary of the invention]
The purpose of this invention is to provide the method that kit that a kind of usefulness contains S100A2 comes detection of lung cancer.
For realizing that a kind of relevant pulmonary cancer diagnosis kit of above-mentioned purpose design is used for the detection method of lung cancer, comprise kit, S100A2 albumen, it is characterized in that this method may further comprise the steps:
(1) the pulmonary cancer diagnosis kit is provided with several containers or liquid chip and label, is respectively equipped with in described container container or the liquid chip
The specific antibody of a, S100A2 albumen; With
B, PBS pH of buffer 7.2-7.4, or 0.01mol sodium citrate buffer solution pH6.0, or 3%H2O2 solution, or horseradish enzyme detectable;
(2) recombinant protein preparation, the nucleotide sequence of amplification S100A2, or with the recombinant expression carrier conversion that contains this nucleosides or transduce to proper host cell;
(3) in proper culture medium, cultivate host cell;
(4) separation, protein purification from nutrient culture media or cell;
(5) perhaps it has antigenic fragment to the S100A2 gene outcome of purifying, is injected in the animal body to induce the generation of polyclonal antibody preparation, the antibody of the S100A2 of specificity combination simultaneously albumen;
(6) SABC: (a) dewaxing and aquation: get paraffin section 30min in xylene of 5 μ m, dewaxing; Alcohol immersion through a series of different stages is to aquation then; (b) antigen retrieval: section is placed 10mM pH6.0 citrate buffer solution (pH6.0), micro-wave oven heating 15min; (c) section after the microwave treatment still places citrate buffer solution 15min, then with the water flushing 5min that flows; (d) section is positioned over 3%H2O2 incubated at room 15min, to eliminate the activity of endogenous peroxydase; (e) section places the S100A2 rabbit polyclonal antibody incubation 60min of 1/500 dilution; (f) DAB (diaminobenzidine) substrate staining through SABC horseradish enzyme system then; (g) haematoxylin redyeing is used in section, and the alcohol immersion through a series of different stages is to xylene then, the section of handling; Microscopy; Replace polyclonal antibody to do negative control with water or damping fluid in the experiment,, accomplish at last and detect with the positive contrast of sample that S100A2 crosses expression.
The coded sequence that recombinant protein prepares S100A2 obtains through RT-PCR; Primer is respectively: S100A2-F:5-GGATCC GAACTTCTGCACAAGG-3 ' and S100A2-R:5 '-CCGCTCGAGAAAGGCATCAACAGTC-3 ', that template is used is cancerous lung tissue cDNA.The dna fragmentation that obtains inserts in the pGEX-4T-1 of BamHI/XohI double digestion construction recombination plasmid pGEX-4T-1-S100A2 behind restriction enzyme BamHI/XohI double digestion.Transform recombinant plasmid to the middle abduction delivering of BL21 (DE3), the S100A2 recombinant protein process purifying of abduction delivering, and through the SDS-PAGE detection.
The polyclonal antibody preparation is with physiological saline dilution S100A2 recombinant protein; Carrying out geometric ratio with incomplete Freund then mixes; Blow and beat to mix fully and form stable emulsion, this emulsion is carried out hypodermic injection carry out intramuscular injection under the skin around the rabbit both shoulders with the back thigh to antigen and adjuvant.Blood sampling 2ml (obtaining the 0.5-1ml preimmune serum) before immune at every turn, and carry out ELISA and Western Blotting detection.If detect negative or antibody titer lower, then need immunity once more.Immunity is 4 times altogether, last bloodletting 20-30ml, and the purifying mixed blood sample, average each purifying can obtain 100-150mg antibody, carries out quality testings such as ELISA and WB at last.
The present invention compares with prior art, and is variant although known S100A2 expresses in some tumour, and its effect in the variety classes tumour also should be different.Therefore, the present invention develops the detectable that can supply clinical practice, with the correlativity of research in depth with extensive checking S100A2 and lung cancer, cancer is diagnosed and better auxiliary treating method is provided for individual patient.
[embodiment]
In conjunction with specific embodiment the present invention is further specified, the manufacturing technology of this device is very clearly concerning this professional people.
Adopt comparison protein group method that the protein expression profile of lung cancer cell line/tissue and normal control cell/tissue is compared analysis, mass spectrum is identified wherein part differential protein point.S100A2 albumen is one of them discrepancy, and promptly it than normal cell high expressed, and can obviously be distinguished in cancer cell.
Adopt the technology of Real-time PCR (hereinafter to be referred as RT-PCR) and SABC; Detected mRNA and the protein expression expression of the S100A2 in the normal tissues of patients with lung cancer and pairing thereof respectively, found that S100A2 expresses the normal tissues apparently higher than its pairing in cancerous lung tissue.This discovery is consistent with the experimental result of comparison protein group.Show that S100A2 can be as a kind of diagnostic tool of lung cancer.
Therefore, design a kind of kit, detected the existence of S100A2 through S100A2 antibody.
Term " S100A2 " is a member in the calbindin S100 gene family; It is positioned between 250～300kb of human chromosome 1q21; And and S100A1, S100A3～10, S100 gene family member such as S100C and epidermal differentiation gene are jointly between the 1750kb zone.
At present, can pass through recombinant DNA technology, express or produce reorganization S100A2 albumen.In general following steps are arranged:
(1) nucleotide sequence of amplification S100A2, or with the recombinant expression carrier conversion that contains this nucleosides or transduce to proper host cell;
(2) in proper culture medium, cultivate host cell;
(3) separation, protein purification from nutrient culture media or cell.
The S100A2 gene outcome of purifying or its have antigenic fragment, and to induce the generation of polyclonal antibody, specificity combines the antibody of S100A2 albumen to injectable simultaneously, also can be business-like antibody in animal body.
SABC specifically comprises: (1) dewaxing and aquation: get paraffin section 30min in xylene of 5 μ m, dewaxing; Alcohol immersion through a series of different stages is to aquation then.(2) antigen retrieval: section is placed 10mM pH6.0 citrate buffer solution (pH6.0), micro-wave oven heating 15min.(3) section after the microwave treatment still places citrate buffer solution 15min, then with the water flushing 5min that flows.(4) section is positioned over 3%H
2O
2Incubated at room 15min is to eliminate the activity of endogenous peroxydase.(5) section places the S100A2 rabbit polyclonal antibody incubation 60min of 1/500 dilution.(6) DAB (diaminobenzidine) substrate staining through SABC horseradish enzyme system then.(7) haematoxylin redyeing is used in section, and the alcohol immersion through a series of different stages is to xylene then.The section of handling, microscopy.Replace polyclonal antibody to do negative control with water or damping fluid in the experiment, cross the positive contrast of sample of expression with S100A2.
Embodiment
The coded sequence that recombinant protein prepares S100A2 obtains through RT-PCR; Primer is respectively: S100A2-F:5 '-GGATCC GAACTTCTGCACAAGG-3 ' and S100A2-R:5 '-CCGCTCGAGAAAGGCATCAACAGTC-3 ', that template is used is cancerous lung tissue cDNA.The dna fragmentation that obtains inserts in the pGEX-4T-1 of BamHI/XohI double digestion construction recombination plasmid pGEX-4T-1-S100A2 behind restriction enzyme BamHI/XohI double digestion.Transform recombinant plasmid to the middle abduction delivering of BL21 (DE3), the S100A2 recombinant protein process purifying of abduction delivering, and through the SDS-PAGE detection.
The Polyclonal Antibody Preparation immunity is with physiological saline dilution S100A2 recombinant protein; Carrying out geometric ratio with incomplete Freund then mixes; Blow and beat to mix fully and form stable emulsion, this emulsion is carried out hypodermic injection carry out intramuscular injection under the skin around the rabbit both shoulders with the back thigh to antigen and adjuvant.Blood sampling 2ml (obtaining the 0.5-1ml preimmune serum) before immune at every turn, and carry out ELISA and Western Blotting detection.If detect negative or antibody titer lower, then need immunity once more.Immunity is 4 times altogether, last bloodletting 20-30ml, and the purifying mixed blood sample, average each purifying can obtain 100-150mg antibody, carries out quality testings such as ELISA and WB at last.
SABC is collected each 20 example of normal tissues section of patients with lung cancer and pairing thereof.(1) dewaxing and aquation: get paraffin section 30min in xylene of 5 μ m, dewaxing; Alcohol immersion through a series of different stages is to aquation then.(2) antigen retrieval: section is placed 10mM pH6.0 citrate buffer solution (pH6.0), micro-wave oven heating 15min.(3) section after the microwave treatment still places citrate buffer solution 15min, then with the water flushing 5min that flows.(4) section is positioned over 3%H
2O
2Incubated at room 15min is to eliminate the activity of endogenous peroxydase.(5) section places the S100A2 rabbit polyclonal antibody incubation 60min of 1/500 dilution.(6) DAB (diaminobenzidine) substrate staining through SABC horseradish enzyme system then.(7) haematoxylin redyeing is used in section, and the alcohol immersion through a series of different stages is to xylene then.The section of handling, microscopy.Replace polyclonal antibody to do negative control with water or damping fluid in the experiment, cross the positive contrast of sample of expression with S100A2.As a result, 16 examples all have high expressed in various degree in the patients with lung cancer, and do not see high expressed in the normal tissues.Therefore, S100A2 high expressed and lung cancer have correlativity closely, can supply the clinical effective ways of cancer being diagnosed and provided for individual patient better supplemental treatment.
Claims (3)
1. a relevant pulmonary cancer diagnosis kit is used for the detection method of lung cancer, comprises kit, S100A2 albumen, it is characterized in that this method may further comprise the steps:
(1) the pulmonary cancer diagnosis kit is provided with several containers or liquid chip and label, is respectively equipped with in described container container or the liquid chip
The specific antibody of a, S100A2 albumen; With
B, PBS pH of buffer 7.2-7.4, or 0.01mol sodium citrate buffer solution pH6.0, or 3%H2O2 solution, or horseradish enzyme detectable;
(2) recombinant protein preparation, the nucleotide sequence of amplification S100A2, or with the recombinant expression carrier conversion that contains this nucleosides or transduce to proper host cell;
(3) in proper culture medium, cultivate host cell;
(4) separation, protein purification from nutrient culture media or cell;
(5) perhaps it has antigenic fragment to the S100A2 gene outcome of purifying, is injected in the animal body to induce the generation of polyclonal antibody preparation, the antibody of the S100A2 of specificity combination simultaneously albumen;
(6) SABC: (a) dewaxing and aquation: get paraffin section 30min in xylene of 5 μ m, dewaxing; Alcohol immersion through a series of different stages is to aquation then; (b) antigen retrieval: section is placed 10mM pH6.0 citrate buffer solution (pH6.0), micro-wave oven heating 15min;
(c) section after the microwave treatment still places citrate buffer solution 15min, then with the water flushing 5min that flows; (d) section is positioned over 3%H2O2 incubated at room 15min, to eliminate the activity of endogenous peroxydase; (e) section places the S100A2 rabbit polyclonal antibody incubation 60min of 1/500 dilution; (f) DAB (diaminobenzidine) substrate staining through SABC horseradish enzyme system then; (g) haematoxylin redyeing is used in section, and the alcohol immersion through a series of different stages is to xylene then, the section of handling; Microscopy; Replace polyclonal antibody to do negative control with water or damping fluid in the experiment,, accomplish at last and detect with the positive contrast of sample that S100A2 crosses expression.
2. a kind of relevant pulmonary cancer diagnosis kit as claimed in claim 1 is used for the detection method of lung cancer; It is characterized in that the coded sequence that recombinant protein prepares S100A2 obtains through RT-PCR; Primer is respectively: S100A2-F:5-GGATCC GAACTTCTGCACAAGG-3 ' and S100A2-R:5 '-CCGCTCGAGAAAGGCATCAACAGTC-3 ', that template is used is cancerous lung tissue cDNA.The dna fragmentation that obtains inserts in the pGEX-4T-1 of BamHI/XohI double digestion construction recombination plasmid pGEX-4T-1-S100A2 behind restriction enzyme BamHI/XohI double digestion.Transform recombinant plasmid to the middle abduction delivering of BL21 (DE3), the S100A2 recombinant protein process purifying of abduction delivering, and through the SDS-PAGE detection.
3. a kind of relevant pulmonary cancer diagnosis kit as claimed in claim 1 is used for the detection method of lung cancer; It is characterized in that the polyclonal antibody preparation is with physiological saline dilution S100A2 recombinant protein; Carrying out geometric ratio with incomplete Freund then mixes; Blow and beat to mix fully and form stable emulsion, this emulsion is carried out hypodermic injection carry out intramuscular injection under the skin around the rabbit both shoulders with the back thigh to antigen and adjuvant.Blood sampling 2ml (obtaining the 0.5-1ml preimmune serum) before immune at every turn, and carry out ELISA and Western Blotting detection.If detect negative or antibody titer lower, then need immunity once more.Immunity is 4 times altogether, last bloodletting 20-30ml, and the purifying mixed blood sample, average each purifying can obtain 100-150mg antibody, carries out quality testings such as ELISA and WB at last.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113075408A (en) * | 2021-03-16 | 2021-07-06 | 上海市同仁医院 | Immunohistochemical quantitative method using histone as internal reference |
| CN113138273A (en) * | 2020-01-17 | 2021-07-20 | 孟民杰 | Kit applied to rapid screening and immune targeted therapy and detection of lung cancer and application thereof |
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2011
- 2011-10-25 CN CN2011103282947A patent/CN102520174A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113138273A (en) * | 2020-01-17 | 2021-07-20 | 孟民杰 | Kit applied to rapid screening and immune targeted therapy and detection of lung cancer and application thereof |
| CN113075408A (en) * | 2021-03-16 | 2021-07-06 | 上海市同仁医院 | Immunohistochemical quantitative method using histone as internal reference |
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