CN101290321A - Membrane associated protein A2 blood serum detection method, detection reagent kit and its uses - Google Patents

Membrane associated protein A2 blood serum detection method, detection reagent kit and its uses Download PDF

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CN101290321A
CN101290321A CNA2008100896574A CN200810089657A CN101290321A CN 101290321 A CN101290321 A CN 101290321A CN A2008100896574 A CNA2008100896574 A CN A2008100896574A CN 200810089657 A CN200810089657 A CN 200810089657A CN 101290321 A CN101290321 A CN 101290321A
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annexin
hcc
biological sample
serum
anxa2
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赵晓航
孙玉琳
乔媛媛
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Cancer Hospital and Institute of CAMS and PUMC
General Hospital of PLA Navy
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PLA NAVY GENERAL HOSIPTAL
Cancer Hospital and Institute of CAMS and PUMC
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

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Abstract

The invention relates to a method for detecting the serum of Annexin A2, a detection kit and an application thereof, in particular to a method for separately using a novel marker Annexin A2 to detect HCC or using the novel marker Annexin A2 together with AFP to detect HCC, the detection kit and the application thereof.

Description

Serum detection method, detection kit and the application thereof of annexin A2
Technical field
The present invention relates to field of biology, use method, detection kit and the application thereof that detects HCC in particular to the new marker detection HCC of independent use or with the AFP coupling.
Background technology
Liver cancer (Liver cancer) is a kind of malignant tumour of serious harm human health, and about 564,000 people of whole world new cases in 2000 ranked fifth the position in all malignant tumours.Since its grade malignancy height, prognosis mala, five year survival rate less than 10%.China is the district occurred frequently of liver cancer, has concentrated the new cases in the whole world about 54%, and wherein (hepatocellular carcinomas HCC) accounts for more than 90% of primary carcinoma of liver to hepatocellular carcinoma [1,2]In the period of 1991-2000, the cause of the death sample survey of China's 169,871 populations shows that its mortality ratio comes the 2nd of whole malignant tumours, and annual death rate is 54.7/100,000 (man 81.2, woman 29.0) [3]
(alpha-fetoprotein AFP) is the HCC diagnosis marker of generally acknowledging clinically at present to alpha-fetoprotein.The 1950's, people such as Bergstrand have found in human foetus's serum that first this electrophoretic mobility is equivalent to the protein of alpha globulin, do not detect its existence in normal adult serum [4]Nineteen sixty, Abelev G and colleague thereof have found this alpha globulin in the hepatocellular carcinoma of mouse, and it is not present in any tissue of normal mouse, verified it be principal ingredient in the tire mouse serum, when the adult rats liver cell regeneration, can reappear [5]Afterwards, the specific alpha globulin of embryo also was detected among HCC patient and the teratoma patients serum [6,7]Therefore, people are with this albumen called after alpha-fetoprotein that mainly is present in fetal tissue.
Known at present, AFP is a kind of secreting type glycoprotein, belongs to the albumin-like gene family with albumin.It mainly is present in rodent and people's serum embryonic period, embryonic phase, synthesizes justacrine in blood by embryonic development yolk-sac entoderm in period and tire liver.The AFP maximum concentration can reach 3-4mg/ml in human embryos, and after the birth, AFP concentration descends rapidly in the serum, and neonatal period is about 10-50 μ g/ml, and normal adult is generally below 10ng/ml.The variation of AFP concentration can be pointed out multiple embryonic development defective in the period of gestation maternal serum [8]Form philtrum, the serum afp of rising does not exist only in patient HCC of 60%-70%, is found in about 20% chronic hepatitis yet, the hepatitis cirrhosis of 20%-60% and some embryonal carcinoma patient [9]In the HCC patient that chronic hepatitis and cirrhosis background are arranged, the diagnostic value of AFP is lower [10,11]Therefore, between 41%-97%, specificity is at 80%-95% for HCC patient's diagnostic sensitivity for AFP, and positive predictive value is paced up and down between 9%-58% [10-12]What is more important has the HCC patients serum AFP level of 20%-30% not raise clinically, thereby has limited its using value in the HCC clinical examination greatly.Therefore, press at present and find and to unite use with AFP, improve the new mark of HCC diagnosis accuracy.
In sum, owing to still lack effective liver cancer detection method at present, therefore, this area presses for exploitation and sets up new liver cancer patient blood serum ELISA detection method and kit that can convenient and swift detection.
For this reason, the inventor has carried out a large amount of R﹠D works, in previous work, the inventor has extracted three hepatoma cell line HepG2, Hep3B and SK-HEP-1, and subcellular components that normal liver cell is HL-7702, and, choose protein spots with difference more than three times through bidimensional electrophoretic separation (2-DE), enzymolysis in the glue is after the evaluation of MALDI-TOF-TOF mass spectrum.In the endochylema component, the inventor has been surprised to find that the protein spots of a specificity overexpression in AFP negative HCC clone SK-HEP-1, through mass spectrum be accredited as annexin 2 (Annexin A2, ANXA2).
ANXA2 belongs to annexins protein family member, is a kind of Ca 2+Ions binding albumen, its C end core domain comprises the binding site of phosphatide, F-actin and heparin, and the N end comprises the phosphorylation regulatory site of PKC (Ser-25) and Src (Tyr-23) [13-15]ANXA2 exists with three kinds of forms in cell: monomer, dimer and the tetramer.Dimer is made up of ANXA2 of a part and the glycerol 3-phosphate acid kinase of a part.The tetramer is made up of bimolecular ANXA2 and bimolecular S100A10 (P11).The tetramer is different along with the type of cell or tissue to the relative quantity of monomer, and the tetramer of ANXA2 approximately is 100% in the intestinal epithelial cell, and the ANXA2 of monomeric form has accounted for more than 50% in the fibroblast of cultivating.Think at present, ANXA2 can intermediary's cell processes such as the vesica transhipment that born of the same parents secrete, endocytosis, actin rely on, pinocytosis, regulate ion channel and influence that cytoskeleton is rebuild, iuntercellular sticks and move, and it is gone back to the nest, implants and keep in the bone marrow microenvironment at candidate stem cell and plays a significant role [16-34]In various transformants, as v-src, v-H-ras, v-mos and SV40 transformant etc., the expression of ANXA2 all can be induced.And its gene expression is subjected to multiple growth factor, as the adjusting of insulin, IGF and EGF.In multiple human tumor as the equal up-regulated of ANXA2 in cancer of pancreas, high differentiation glioma, cancer of the stomach, clear-cell carcinoma, lung cancer, HCC and the acute promyelocytic leukemia, but in prostate cancer down-regulated expression [35-42]Therefore show that the rise of ANXA2 is relevant with cell conversion process.In addition, approximately the interior ANXA2 of the cell of 10%-15% is relevant with nucleus.ANXA2 can combine with dna sequence dna, is the constituent that stimulates the primer recognition complex of archaeal dna polymerase alpha active [43-46]ANXA2 has the RNA binding ability, is the constituent of mRNP in the body, has been found that at present ANXA2 can be directly and 3 '-UTR of c-Myc mRNA interaction, and the transhipment that it is special also navigates to cytoskeleton in the endochylema.After cell is crossed expression ANXA2, cause the c-Myc protein expression level to raise simultaneously [47,48]Result of study in breast cancer and head and neck neoplasm finds that ANXA2 is potential plasminogen and histiotype plasminogen activator protein (t-PA) acceptor, may participate in tumor invasion and transfer process [49,50]
People such as Dreier R once utilized immunohistochemical staining to study the expression of Annexin protein family member in each normal organ of human body, tissue, found that liver cell and the Kupffer cell in the liver all do not expressed Annexin A2 [51]People such as Tucker CJ have studied HCC that arsenic acid brings out and the difference expression gene spectrum between the contrast normal liver in animal model, a difference expression gene that wherein identifies is exactly Annexin A2, it obviously raises in tumor tissues, real time PCR found that, in normal control bull C3H mouse, the expression of ANXA2 is 1.0 ± 0.3, and the expression of Annexin A2 raises 9 times and 49 times in other normal structure of the mouse cancer of bringing out HCC and tumor tissues [52]People such as Lim SO utilize the 2-DE-MAILDI-TOF technical tactic to contrast the difference of normal, cirrhosis and hepatocellular carcinoma histone express spectra, one of differential protein that identifies is exactly Annexin A2, and the expression of this albumen in normal and cirrhotic tissue is starkly lower than the HCC tumor tissues [53]But, these two pieces of researchs only from the angle examination of genome and protein group the differential expression between HCC and the healthy liver, on clinical sample, Annexin A2 is not carried out any checking, and, can in human serum, detect without any Annexin A2 at present, and the report relevant with human diseases.
Summary of the invention
In view of the vital role of ANXA2 in tumor development, the inventor has carried out the check analysis of large sample amount to its expression in liver cancer tissue, the result shows, in the Western blot result of 19 routine paired liver cancer tissues, the up-regulated rate of ANXA2 in tumor tissues is 68.4 (13/19), and the immunohistochemical staining result of 40 example pairing HCC tissues shows, its The positive expression rate in tumor tissues is 60% (24/40), and the The positive expression rate in the other normal structure of cancer only is 10%, through the Chi-square testing identity, ANXA2 be a kind of in liver cancer tissue the protein (p＜0.0001) of high expressed.Therefore, the inventor thinks that the expression of ANXA2 albumen and liver cancer have crucial the contact, can be used as the responsive marker protein of liver cancer relative specificity.Though ANXA2 does not have signal peptide,, bibliographical information, it is present in the ectosome (exosome), and we infer that it can be secreted into blood through non-classical secretory pathway [54,55], in liver cancer patient blood serum/blood plasma, can detect, and learn the specificity marker thing that detects as the liver cancer early stage blood serum.On the basis of above-mentioned discovery, the inventor has set up the indirectly two sandwich ELISA detection methods of serum of human ANXA2 first, and its testing result is estimated.And then the invention provides serum ELISA detection method, detection kit and the application thereof of the new mark AnnexinA2 that improves the HCC diagnosis accuracy.Particularly, the present invention includes the following aspects.
On the one hand, the invention provides the method for following acquisition as the information of intermediate result, whether described method is unusual for the Annexin A2 level that detects in the biological sample to be measured, and then obtains the method as the information of intermediate result, and described method comprises step:
1) the Annexin A2 level in the mensuration biological sample to be checked;
2) the Annexin A2 level of Annexin A2 level in the biological sample more to be checked and normal biological sample contrast;
3) obtain information as intermediate result,
4) determine according to comparative result whether biological sample to be checked might be the HCC sample.
2. above-mentioned 1 method further comprises the joint-detection with AFP.
3. above-mentioned 1 or 2 method, wherein the biological sample of step 1) is tissue, body fluid or the excreta that has broken away from human body or animal body.
4. above-mentioned 1 or 2 method, wherein the biological sample of step 1) is a serum.
5. above-mentioned 1 or 2 method, wherein the mensuration of step 1) is serum ELISA method.
6. use Annexin A2 to detect the method for HCC, described method comprises step:
1) the Annexin A2 level in the mensuration biological sample to be checked;
2) the Annexin A2 level of Annexin A2 level in the biological sample more to be checked and normal biological sample contrast;
3) determine according to comparative result whether biological sample to be checked is the HCC sample.
7. above-mentioned 6 method further comprises the joint-detection with AFP.
8. above-mentioned 6 method, wherein the biological sample of step 1) is a serum.
9. above-mentioned 6 method, wherein the mensuration of step 1) is serum ELISA method.
10.Annexin A2 is as the purposes of the mark of HCC.
11. above-mentioned 10 purposes, wherein Annexin A2 and AFP use in conjunction.
12. be used to detect the HCC detection kit of Annexin A2.
13. above-mentioned 12 HCC detection kit, comprising: as the Annexin A2 of standard, anti-Annexin A2 antibody.
14. be used for the HCC detection kit of joint-detection Annexin A2 and AFP.
Description of drawings:
Fig. 1 is experimenter's detection curve that Serum AFP, ANXA2 and two indexs are united use.Dotted line is represented AFP among the figure, and pecked line is represented ANXA2, and on behalf of two indexs, solid line unite use.A represents area under curve.
Embodiment
In order further clearly to set forth the present invention, provide specific embodiments of the invention below, but content of the present invention is not limited to described embodiment, the distortion of the method for any this law and the equivalence of product and modification etc. all comprise within the scope of the invention.
Embodiment 1: the indirectly two sandwich ELISA detection methods of serum that adopt human ANXA2
For biological sample is detected, carried out following ELISA detection method, the concrete steps of described method are:
1. bag quilt: with 50mM carbonate buffer solution (the 15mM Na of pH 9.6 2CO 335mMNaHCO 3) as dilution, (Santa CruzBiotechnology company, Cat.No.sc-1924) dilution is 2 μ g/ml with the anti-people ANXA2 of goat antibody.In the micropore of 96 hole ELISA Plate of polystyrene, add 50 μ l coating buffers, seal with preservative film, in 4 ℃ of refrigerators, place and spend the night (＞16h).Next day is with PBST damping fluid (137mM NaCl, the 2mM KH of 350 μ l 150mM 2PO 4, 10mM Na 2HPO 4, add 0.05% Tween-20, being adjusted to pH7.4) and immersion type washes plate, 3min * 3 time.
2. sealing: every hole add 300 μ l 2%BSA (Sigma-Aldrich company, Cat.No.A7906), room temperature sealing 4h.Wash plate with 350 μ l PBST damping fluid immersion types, 1min * 3 time.
3. antigen is hatched: the people who adds the HCC patients serum (diluting at 1: 10 with the PBST damping fluid) of 50 μ l or gradient dilution in each reacting hole ANXA2 albumen (the Abnova company that recombinates, Cat.No.H00000302-P02), hatch 1h for 37 ℃, simultaneously, substitute antigen as negative control with lavation buffer solution.Each sample is provided with 3 parallel holes.After hatching end, discard antigen, wash plate, 3min * 3 time with 350 μ l PBST damping fluid immersion types.
4. detection antigen-reactive: (Santa Cruz Biotechnology company is Cat.No.sc-28385) as detecting antibody, incubated at room 1h with the mouse anti human ANXA2 antibody of 1 μ g/ml of PBST damping fluid dilution to add 50 μ l in each reacting hole.Then discard, wash plate, 3min * 3 time with 350 μ l PBST damping fluid immersion types.
5. two anti-hatching: in each reacting hole, add 50 μ l with the goat anti-mouse IgG of the horseradish peroxidase-labeled of PBST damping fluid dilution in 1: 5000 (JacksonImmunoReasearch company, Cat.No.115-005-003), incubated at room 1h.Then discard, wash plate, 3min * 3 time with 350 μ l PBST damping fluid immersion types.
6.TMB the colour developing of (3,3 ', 5,5 '-tetramethyl benzidine) substrate: (Sigma-Aldrich company Cat.No.15053) is dissolved in the 5ml absolute ethyl alcohol, is configured to the TMB stock solution to take by weighing 10mg TMB.Then get 0.5ml TMB stock solution and add 10ml phosphoric acid citric acid substrate buffer solution (51.4mM Na 2HPO 4, the 24.3mM citric acid, pH5.0) in, and add the H of 32 μ l0.75% 2O 2Mixing is configured to the TMB chromophoric solution.In each reacting hole, add 100 μ l TMB chromophoric solutions, mixing gently, incubated at room 30min.
7. stop: after treating that color is satisfied and stable, every hole adds 100 μ l 2M H 2SO 4Solution cessation reaction, and mixing gently.
8.OD the mensuration of value: immediately ELISA Plate is placed Model 680 microplate reader (Bio-Rad company), OD450/570 dual wavelength reading.
9. result's interpretation: at first determine the reading of 3 negative control holes, average as the background reading.
Then for each routine blood serum sample or reorganization ANXA2 standard items, calculate the coefficient of variation (CV) between the hole of 3 parallel holes earlier, less than 15% sample, get the OD value of the mean value of 3 repetitions for those CV values as this sample; The CV value is greater than 15% sample, after removing an exceptional value, recomputate variation between the hole (poor/two hole OD value sums of variation=two hole OD values between the hole), if should variation value＜15%, think that this sample is effective case, and get the OD value of two hole mean values as this sample, otherwise, this example sample given up.Then, utilize the reorganization ANXA2 standard items drawing standard curve of gradient dilution, thereby calculate the effectively serum ANXA2 concentration of case of each example.Annotate: above-mentioned experimental technique material therefor except that special indicating, is homemade analytical reagent.
Embodiment 2: the clinical detection of the indirectly two sandwich ELISA detection methods of the serum of human ANXA2 is used
Utilize the said method of embodiment 1, the 30 routine health adults that age, sex are complementary (51 years old mean age, maximum 70 years old age, minimal ages 33 years old) and 98 routine HCC patient (54 years old mean age, maximum 83 years old age, minimal ages 15 years old) serum ANXA2 concentration is measured.The OD value of the concentration gradient of test findings and typical curve and deduction background sees Table 1, and both linearly dependent coefficients are 0.971, show that this concentration gradient drops in the range of linearity of detection reaction substantially.Show that equally this systematic comparison of being set up is stable, all effective to all case results, the CV value of most samples is controlled in 10%.
The setting of table 1:ANXA2 typical curve and the OD value of detection
Figure A20081008965700101
Experimental result finds that the concentration of the serum ANXA2 of health adult is 17.58 μ g/ml, is 4.81～29.29 μ g/ml between detection zone.And HCC patient's serum ANXA2 mean concentration is 28.68 μ g/ml, and minimum value is 13.45, and maximal value is 230.66 μ g/ml.Through Mann-Whitney Rank Sum check, both differences have conspicuousness (p＜0.0001).Simultaneously, do not have between the serum ANXA2 concentration in the normal healthy controls and its age, the sex tangible correlativity (p=0.9462, p=0.3646).The HCC patient that AFP is positive and negative, serum ANXA2 expression does not have notable difference (p=0.1595).And we find that low differentiation HCC patient's serum ANXA2 is apparently higher than middle poorly-differentiated cases (p＜0.05).
Embodiment 3: the parallel detection of adopting contrast method to carry out
Utilize electrochemical luminescence method (the conventional clinically method of using) to measure the serum concentration of AFP of all ELISA samples of previous embodiment 2, the result shows that health adult and HCC patient's Serum AFP mean concentration is respectively 3.5875ng/ml and 31.415ng/ml.Draw these two albumen respectively and be used for experimenter's curve (ROC curve) that HCC detects (Fig. 1), found that the area under curve of AFP is 0.81, the area under curve of ANXA2 is 0.78, the diagnostic value of these two albumen basic identical (Chi-square Test, p=0.6096).At this moment, the serum concentration of AFP that obtains the excellent diagnostics effect is 11ng/ml, and diagnostic sensitivity is 93.3%, and specificity is 63.5%.And the threshold value of ANXA2 is 27.93 μ g/ml, and diagnostic sensitivity and the specificity of this moment are respectively 96.7% and 52.1%.When uniting these two serological index of use, the ROC area under curve is increased to 0.87, and diagnostic sensitivity and specificity are increased to 100% and 72.9%.
The above results shows, the serum ANXA2 ELISA detection method of being set up has good reproducibility, highly sensitive characteristics.The serum ANXA2 that uses it for 128 routine health adults and HCC patient measures and finds that this index has very high detection sensitivity, shows that it may be a very potential serum early screening index.And, the diagnosis of itself and AFP is renderd a service basic identical, particularly, use ANXA2 independently to detect separately to HCC, it has approximate diagnostic value with present clinical AFP commonly used, and sensitive more (diagnostic sensitivity of ANXA2 is 96.7%, and AFP is 93.3%), be particularly suitable for crowd's examination of tumour.Use these two serological index if unite, then can 100% detect HCC patient.
Embodiment 4: the liver cancer detection kit that detects ANXA2
The present invention also provides a kind of liver cancer detection kit, and the people ANXA2 that wherein contains reorganization is as protein standard, contain anti-people ANXA2 antibody (coated antibody: working concentration 2 μ g/ml, dilution buffer liquid is the 50mM carbonate buffer solution, pH 9.0; Detect antibody: working concentration 1 μ g/ml, dilution buffer liquid is the PBST damping fluid of 150mM, pH 7.4), and other detectable among the embodiment 1.
Use kit of the present invention to adopt the method for embodiment 1 to finish clinical testing 128 examples, detecting positive rate is 100%, and sensitivity is up to 96.7%.According to literature search, still there is not the report of identical work at present both at home and abroad.
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Claims (14)

1. whether the Annexin A2 level that detects in the biological sample to be measured is unusual, and then obtains the method as the information of intermediate result, and described method comprises step:
1) the Annexin A2 level in the mensuration biological sample to be checked;
2) the Annexin A2 level of Annexin A2 level in the biological sample more to be checked and normal biological sample contrast;
3) obtain information as intermediate result,
4) determine according to comparative result whether biological sample to be checked might be the HCC sample.
2. the method for claim 1 further comprises the joint-detection with AFP.
3. claim 1 or 2 method, wherein the biological sample of step 1) is tissue, body fluid or the excreta that has broken away from human body or animal body.
4. claim 1 or 2 method, wherein the biological sample of step 1) is a serum.
5. claim 1 or 2 method, wherein the mensuration of step 1) is serum ELISA method.
6. use Annexin A2 to detect the method for HCC, described method comprises step:
1) the Annexin A2 level in the mensuration biological sample to be checked;
2) the Annexin A2 level of Annexin A2 level in the biological sample more to be checked and normal biological sample contrast;
3) determine according to comparative result whether biological sample to be checked is the HCC sample.
7. the method for claim 6 further comprises the joint-detection with AFP.
8. the method for claim 6, wherein the biological sample of step 1) is a serum.
9. the method for claim 6, wherein the mensuration of step 1) is serum ELISA method.
10.Annexin A2 is as the purposes of the mark of HCC.
11. the purposes of claim 10, wherein Annexin A2 and AFP use in conjunction.
12. be used to detect the HCC detection kit of Annexin A2.
13. the HCC detection kit of claim 12, comprising: as the Annexin A2 of standard, anti-Annexin A2 antibody.
14. be used for the HCC detection kit of joint-detection Annexin A2 and AFP.
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