CN101760557A - Reagent kit for auxiliary diagnosis of hepatoma - Google Patents
Reagent kit for auxiliary diagnosis of hepatoma Download PDFInfo
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- CN101760557A CN101760557A CN201010118539A CN201010118539A CN101760557A CN 101760557 A CN101760557 A CN 101760557A CN 201010118539 A CN201010118539 A CN 201010118539A CN 201010118539 A CN201010118539 A CN 201010118539A CN 101760557 A CN101760557 A CN 101760557A
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- 206010073071 hepatocellular carcinoma Diseases 0.000 title claims abstract description 39
- 238000003745 diagnosis Methods 0.000 title claims abstract description 28
- 239000003153 chemical reaction reagent Substances 0.000 title abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 230000014509 gene expression Effects 0.000 claims abstract description 11
- 239000002299 complementary DNA Substances 0.000 claims abstract description 5
- 238000012360 testing method Methods 0.000 claims description 19
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- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
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- 238000003149 assay kit Methods 0.000 claims 1
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- 210000004369 blood Anatomy 0.000 abstract 3
- 238000012546 transfer Methods 0.000 description 13
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- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 9
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- 206010027476 Metastases Diseases 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
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- 241000282414 Homo sapiens Species 0.000 description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a reagent kit for auxiliary diagnosis of hepatoma. The reagent kit provided by the invention comprises a compound for detecting Rab27B gene expression. The application of the compound for detecting the Rab27B gene expression in preparing the reagent kit for the auxiliary diagnosis of the hepatoma also belongs to the protection range of the invention. The compound for detecting the Rab27B gene expression is 1 or 2 as follows: 1. a PCR primer of a cDNA for detecting an Rab27B gene; 2. a monoclonal antibody or a polyclonal antibody for detecting Rab27B albumen. Experiments prove that the concentration mean value of the Rab27B albumen of an LMS group (no metastatic group recurs within two years when in blood collection) is 2.08ng/mul, the concentration mean value of the Rab27B albumen of an HMS group (a metastatic group occurs within one year when in blood collection) is 7.03ng/mul, the concentration mean value of the Rab27B albumen of an AMS group (the metastatic group occurs when in blood collection) is 17.80ng/mul, and the albumen contents among the three groups have marked differences.
Description
Technical field
The present invention relates to a kind of test kit of auxiliary diagnosis of hepatoma.
Background technology
(hepatocellular carcinoma HCC) is a kind of common, malignant tumour that lethality rate is high to hepatocellular carcinoma, and annual about 650,000 people die from HCC, and its sickness rate has the trend that increases.This disease is mainly in South East Asia and Africa, wherein has approximately to betide China more than 50%, occupies second of China's cancer mortality.The HCC early diagnosis is difficult, and disease progression is fast, and prognostic level is low.Excision is considered to the best means that HCC obtains radical cure always, but only has the patient of 10-20% to be fit to intervene by performing the operation; In addition, even radical excision still has the patient of 60-70% transfer and relapse can occur and threat to life in 5 years, the transfer and relapse rate of topical therapeutic is then higher.The transfer of HCC is the biggest obstacle that influences result of treatment and prognosis, is the major cause that causes its high mortality.
In recent years, carried out big quantity research and found many important molecules at the transfer of HCC, as: the regulatory factor of adhesion molecule, cytoskeleton and adjusting albumen, extracellular matrix degrading enzyme, oncogene and cancer suppressor gene, vasculogenesis, tumour immunity associated molecule etc.Also be HCC transfer process and tumour cell to stick motion, cell proliferation, extracellular matrix, immunity of organism, tumor-blood-vessel growth etc. multifactor relevant.Although to the big quantity research of HCC transfer having carried out, the molecular mechanism of HCC transfer at present is still indeterminate, finds to predict the molecular marker of HCC relapse and metastasis as yet and effectively treat target.
Alpha-fetoprotein (alpha fetoprotein, AFP) be present the most frequently used HCC diagnosis marker, but AFP is still undesirable as HCC examination at present and topmost biomarker of state of illness monitoring, and its diagnostic sensitivity, specificity and positive prediction rate have only 39%-65%, 76%-94% and 9%-50% respectively.Even the AFP positive can not judge that HCC has or not transfer.Except that AFP, also have AFP-L3 (AFP heteroplasmon 3), DCP (γ-decarboxylation thrombogen), IGF-1 (insulin-like growth factor-i) according to what U.S. FDA regulation entered that II, III phase study, the research report is arranged in addition but further research verify also have tens of kinds of marks.But these marks are all very limited aspect the diagnosis sensitivity of HCC and specificity, can not satisfy particularly that HCC is early stage, the needs of transfer and relapse diagnosis.
Summary of the invention
The object of the present invention is to provide the transcellular test kit of a kind of auxiliary diagnosis of hepatoma or auxiliary diagnosis of hepatoma.
Test kit provided by the invention comprises the used compound of detection Rab27B genetic expression.
The used compound of above-mentioned detection Rab27B genetic expression is following 1) or 2):
1) the PCR primer of the cDNA of detection Rab27B gene;
2) detect proteic monoclonal antibody of Rab27B or polyclonal antibody.
Proteic monoclonal antibody of above-mentioned detection Rab27B or polyclonal antibody all can obtain from commercial channels.
The proteic aminoacid sequence of above-mentioned Rab27B is shown in sequence in the sequence table 2; Described Rab27B gene is the proteic encoding gene of described Rab27B, preferably the gene shown in the sequence 1 in the sequence table.
The mentioned reagent box also comprises the colour developing liquid that enzyme-linked immunosorbent assay (ELISA) is used.
The mentioned reagent box also comprises the used reaction terminating liquid of enzyme-linked immunosorbent assay (ELISA).
The above-mentioned used compound of detection Rab27B genetic expression is being prepared as follows 1) or 2) test kit in application also belong within protection scope of the present invention:
1) test kit of auxiliary diagnosis of hepatoma;
2) the transcellular test kit of auxiliary diagnosis of hepatoma.
The used compound of above-mentioned detection Rab27B genetic expression is following 1) or 2):
1) the PCR primer of the cDNA of detection Rab27B gene;
2) detect proteic monoclonal antibody of Rab27B or polyclonal antibody.
Proteic monoclonal antibody of above-mentioned detection Rab27B or polyclonal antibody all can obtain from commercial channels.
The proteic aminoacid sequence of above-mentioned Rab27B is shown in sequence in the sequence table 2; Described Rab27B gene is the proteic encoding gene of described Rab27B, preferably the gene shown in the sequence 1 in the sequence table.
The mentioned reagent box also comprises the colour developing liquid that enzyme-linked immunosorbent assay (ELISA) is used.
The mentioned reagent box also comprises the used reaction terminating liquid of enzyme-linked immunosorbent assay (ELISA).
Experimental results show that: the proteic concentration mean value of LMS group (not having the relapse and metastasis group during blood sampling in two years) Rab27B is 2.08ng/ μ l, HMS group (the transfer group takes place in 1 year during blood sampling) is 7.03ng/ μ l, AMS group (the transfer group has taken place during blood sampling) is 17.80ng/ μ l, the protein content significant difference between three groups.By the ROC tracing analysis, as negative case, AMS group and HMS group calculate diagnosis as positive case best operating point is 97.06% for the diagnosis specific degree with the LMS group, and sensitivity is 63.64%; LMS and HMS are organized as negative case, the AMS group calculates diagnosis as positive case best operating point is 98.53% for the diagnosis specific degree, sensitivity is 34.38%, so Rab27B can be used as the potential mark whether diagnosing hepatocellular carcinoma has shifted.
Description of drawings
Fig. 1 is the protein content of Rab27B among the different HCC patients serums.
Fig. 2 is the diagnostic value of ROC tracing analysis Rab27B, and (A) ROC analyzes Rab27B diagnosis metastatic potential: LMS group as negative case, and HMS and AMS group are as positive case; (B) whether ROC analysis Rab27B diagnosis is shifted: HMS and LMS group are as negative case, and AMS organizes as positive case.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.
Among the following embodiment, if no special instructions, be ordinary method.
The protein content of Rab27B among embodiment 1, the different hepatocellular carcinoma patients serums of detection
Present embodiment uses be enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA).
1, the making of typical curve
Get the Rab27B standard protein respectively (available from Santa Cruz company, article No. sc-22993p) 0ng, 0.01ng, 0.02ng, 0.05ng, 0.1ng, 0.2ng, 0.5ng, 1ng, 1.5ng and 2ng coated elisa plate, each gradient is done 2 parallel holes, after 4 ℃ of overnight incubation, and washing lotion washing 3 times; Every hole adds bovine serum albumin (Jackson company) (BSA-IgG free) sealing of 200 μ l 3%, hatches 2h for 37 ℃, washs 3 times; Every hole adds Rab27B and resists (available from Santa Cruz company, article No. sc-22993) (1: 1000PBS-BT dilutes) 100 μ l more, after 4 ℃ of overnight incubation, washs 3 times; Every hole adds the anti-sheep two of HRP-donkey anti-(Jackson company) (PBS-BT dilution in 1: 7500) 100 μ l, hatches 1h for 37 ℃, washs 3 times; Add A, B colour developing liquid (Beijing health is century) (1: 19) 100 μ l/ holes, 37 ℃ of colour developings are to blue; Add stop buffer 80 μ l/ holes, the 450nm wavelength is measured the OD value; The drawing standard curve.
2, hepatocellular carcinoma patients serum sample
Hepatocellular carcinoma patients serum collects from clinical tumor hospital of Peking University, and the age is 40 to 70 years old, wherein LMS group (not having the relapse and metastasis group during blood sampling in two years) 30 examples; HMS group (the transfer group takes place in 1 year during blood sampling) 30 examples; AMS organizes (the transfer group has taken place during blood sampling) 30 examples.Equal underwent operative of all cases and pathologic finding are made a definite diagnosis.
3, measure the content (Fig. 1) of Rab27B in patients serum's sample
Serum sample weaker concn (1: 3500), 3 groups of every holes of the every routine sample of patients serum add 100 μ l wrapper sheets, do a parallel multiple hole, after 4 ℃ of overnight incubation, washing lotion washing 3 times; Every hole adds bovine serum albumin (BSA-IgG free) sealing of 200 μ l3%, hatches 2h for 37 ℃, washs 3 times; Every hole adds Rab27B and resists (1: 1000PBS-BT dilutes) 100 μ l more, after 4 ℃ of overnight incubation, washs 3 times; Every hole adds the anti-sheep two of HRP-donkey anti-(1: 7500PBS-BT dilutes) 100 μ l, hatches 1h for 37 ℃, washs 3 times; Add A, B colour developing liquid (1: 19) 100 μ l/ holes, 37 ℃ of colour developings are to blue; Add stop buffer 80 μ l/ holes, the 450nm wavelength is measured the OD value; According to OD value separately, calculate the content of Rab27B in each serum sample.The result as shown in Figure 1, according to the proteic typical curve of resulting Rab27B and respectively organize the measured OD value of patients serum's sample, calculate gained and respectively organize the proteic content of Rab27B in patients serum's sample, wherein the proteic concentration mean value of LMS group Rab27B is 2.08ng/ μ l, the HMS group is 7.03ng/ μ l, the AMS group is 17.80ng/ μ l, Rab27B protein content in each group sample is carried out statistical analysis, the content difference of finding the target protein between each group has significant statistical significance, also is significant difference between each group.
4, statistical analysis (Fig. 2)
By Excel software Rab27B protein content in each group patients serum sample is carried out the statistics descriptive analysis, to passing through the difference between each group of one-way analysis of variance between each group; Draw the differential diagnosis value that ROC tracing analysis Rab27B shifts for hepatocellular carcinoma by GraphPad Prism software.The result is as shown in Figure 2: A figure with the LMS group as negative case, AMS group and HMS group are as positive case, analytical results is as can be known: area under curve is 0.9011, the best operating point that (sensitivity/1-specific degree) calculates diagnosis according to positive likelihood ratio is 97.06% for the diagnosis specific degree, sensitivity is 63.64%, so Rab27B can be used as the potential mark that diagnosing hepatocellular carcinoma shifts.B figure organizes LMS and HMS as negative case, the AMS group is as positive case, analytical results is as can be known: area under curve is 0.9344, the best operating point that (sensitivity/1-specific degree) calculates diagnosis according to positive likelihood ratio is 98.53% for the diagnosis specific degree, sensitivity is 34.38%, so Rab27B can be used as the potential mark whether diagnosing hepatocellular carcinoma has shifted.
Sequence table
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Claims (10)
1. detect the used compound of Rab27B genetic expression and be prepared as follows 1) or 2) test kit in application:
1) test kit of auxiliary diagnosis of hepatoma;
2) the transcellular test kit of auxiliary diagnosis of hepatoma.
2. application as claimed in claim 1 is characterized in that: the used compound of described detection Rab27B genetic expression is following 1) or 2):
1) the PCR primer of the cDNA of detection Rab27B gene;
2) detect proteic monoclonal antibody of Rab27B or polyclonal antibody.
3. application as claimed in claim 1 or 2 is characterized in that: the proteic aminoacid sequence of described Rab27B is shown in sequence in the sequence table 2; Described Rab27B gene is the proteic encoding gene of described Rab27B, preferably the gene shown in the sequence 1 in the sequence table.
4. application as claimed in claim 3 is characterized in that: described test kit also comprises the colour developing liquid that enzyme-linked immunosorbent assay is used.
5. as claim 3 or 4 described application, it is characterized in that: described test kit also comprises the reaction terminating liquid that enzyme-linked immunosorbent assay is used.
6. auxiliary diagnosis of hepatoma or the transcellular test kit of auxiliary diagnosis of hepatoma comprise and detect the used compound of Rab27B genetic expression.
7. test kit as claimed in claim 6 is characterized in that: the used compound of described detection Rab27B genetic expression is following 1) or 2):
1) the PCR primer of the cDNA of detection Rab27B gene;
2) detect proteic monoclonal antibody of Rab27B or polyclonal antibody.
8. as claim 6 or 7 described test kits, it is characterized in that: the proteic aminoacid sequence of described Rab27B is shown in sequence in the sequence table 2; Described Rab27B gene is the proteic encoding gene of described Rab27B, preferably the gene shown in the sequence 1 in the sequence table.
9. test kit as claimed in claim 8 is characterized in that: described test kit also comprises the colour developing liquid that enzyme-linked immunosorbent assay is used.
10. test kit as claimed in claim 8 or 9, it is characterized in that: described test kit also comprises the reaction terminating liquid that enzyme-linked immunosorbent assay is used.
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| CN 201010118539 CN101760557B (en) | 2010-03-04 | 2010-03-04 | Reagent kit for auxiliary diagnosis of hepatoma |
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| CN 201010118539 CN101760557B (en) | 2010-03-04 | 2010-03-04 | Reagent kit for auxiliary diagnosis of hepatoma |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323601A (en) * | 2012-03-22 | 2013-09-25 | 北京蛋白质组研究中心 | Application of S100A9 protein detection substance in hepatocellular carcinoma screening kit preparation |
| CN104535766A (en) * | 2014-10-30 | 2015-04-22 | 中国人民解放军总医院第一附属医院 | Peripheral blood exosome-sourced liver cancer diagnosis and prognosis marker and applications thereof |
| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
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2010
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| AU2004248120A1 (en) * | 2003-05-28 | 2004-12-23 | Genomic Health, Inc. | Gene expression markers for predicting response to chemotherapy |
| AU2005323173A1 (en) * | 2004-12-01 | 2006-07-13 | The Curators Of The University Of Missouri | Modulator of alpha-synuclein toxicity |
| AU2007210159A1 (en) * | 2006-01-26 | 2007-08-09 | Foldrx Pharmaceuticals, Inc. | Compounds and methods for modulating protein trafficking |
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Non-Patent Citations (1)
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| YANRU CHEN 等: "Rab27b is associated with fusiform vesicles and may", 《PNAS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103323601A (en) * | 2012-03-22 | 2013-09-25 | 北京蛋白质组研究中心 | Application of S100A9 protein detection substance in hepatocellular carcinoma screening kit preparation |
| CN103323601B (en) * | 2012-03-22 | 2016-04-20 | 北京蛋白质组研究中心 | The application of S100A9 Protein Detection thing in preparation examination hepatocellular carcinoma kit |
| CN104535766A (en) * | 2014-10-30 | 2015-04-22 | 中国人民解放军总医院第一附属医院 | Peripheral blood exosome-sourced liver cancer diagnosis and prognosis marker and applications thereof |
| CN104535766B (en) * | 2014-10-30 | 2016-04-13 | 中国人民解放军总医院第一附属医院 | The diagnosing cancer of liver that peripheral blood exosome originates and prognostic marker and application thereof |
| CN105388302A (en) * | 2015-12-22 | 2016-03-09 | 中国医学科学院输血研究所 | Detection method for content of Tau protein antibodies in human immune globulin product |
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| CN101760557B (en) | 2013-08-14 |
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