CN111239389A - Autoantibody marker for distinguishing liver cell liver cancer from normal person and screening method thereof - Google Patents
Autoantibody marker for distinguishing liver cell liver cancer from normal person and screening method thereof Download PDFInfo
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- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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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
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Abstract
An autoantibody marker for distinguishing liver cell cancer from normal human and a screening method thereof. The invention adopts a protein small chip (HCC Focused array) containing 100 recombinant proteins for liver cancer focusing to carry out the control detection of liver cells of liver cancer patients and healthy people, and determines 16 candidate autoantibody markers with certain distinguishing capability or combined distinguishing capability through response data comparison and analysis. The invention is helpful to change the current situation of liver cancer diagnosis and curative effect monitoring, and has important clinical value for liver cancer diagnosis.
Description
Technical Field
The present invention relates to the field of biomedicine, in connection with applied basic medical research and clinical research; oncology, diagnostics and immunology are contemplated.
Background
Hepatocellular carcinoma (HCC for short) is one of common malignant tumors, has hidden onset, high malignancy and high fatality rate, so that timely and accurate diagnosis is very important for improving the survival rate of patients. At present, the early diagnosis of liver cancer is mainly carried out clinically by combining the imaging and pathological examination of Alpha Fetoprotein (AFP); however, AFP is not ideal for the specificity and sensitivity of liver cancer screening. With the development of molecular biology, a variety of new markers have been discovered in succession, particularly for the diagnostic value of autoantibodies.
The autoantibody is a specific antibody generated by an organism to generate immune response to a tumor abnormal antigen, and has the following characteristics: (1) the generation process of the accompanying tumor is prior to clinical symptoms, and the method is suitable for early diagnosis; (2) the production of autoantibodies is the result of an immune reaction, and antibodies are more easily detected than proteins by amplification of the immune system; (3) specific for a particular type of tumor. The existence of some antigens is found to be specific to tumors, but no specificity exists among different tumors, such as p 53; there are also some antigens that are specific to a particular tumor, for example many antigens of prostate cancer are oxidative stress related proteins.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a candidate autoantibody marker for distinguishing liver cell liver cancer from normal people and a screening method thereof so as to change the current situation of liver cancer diagnosis and curative effect monitoring.
The candidate autoantibody marker has distinguishing capacity or combined distinguishing capacity and specifically comprises NPM1, DCAF4L2, TMOD1, CIAPN1, KDM1A, ANKRD13D, ZNF428, CA12, MAS1, C1QTNF3, ASAH1, MS4A3, WTAP, DAB1, SLC44A3 and EGFR.
The invention also provides a screening method of the candidate autoantibody marker for distinguishing the liver cancer of the liver cell from the normal person, which comprises the following steps: and detecting the liver cell liver cancer patient and healthy person contrast by adopting a protein chip containing 100 recombinant proteins and focusing on the liver cell liver cancer, and determining the candidate autoantibody marker by responding data comparison and analysis.
Further, the screening method of the candidate autoantibody marker for distinguishing the liver cancer of the liver cell from the normal human of the invention specifically comprises the following steps:
1) rewarming;
2) and (3) sealing: fixing 14blocks of fences on the rewarming chip, adding sealing liquid into each block after the blocks are fixed, placing the blocks on a side swing table, and sealing at room temperature.
3) Incubation of serum samples: after the sealing is finished, pouring the sealing liquid completely, then quickly adding the prepared serum incubation liquid, laterally swinging a shaker, and incubating overnight;
4) primary cleaning: taking out the chip and the chip clamp together, sucking out the sample, then quickly adding PBST with the same volume, and repeating the steps for a plurality of times to ensure that no cross contamination exists among serum samples when the chip clamp is detached; after the chip clamp is removed, the chip is placed in a chip cleaning box with cleaning fluid, and is cleaned by a horizontal shaking table at room temperature;
5) and (3) secondary antibody incubation: transferring the chip to an incubation box added with a secondary antibody incubation solution, laterally swinging a table, keeping out of the sun, and keeping at room temperature;
6) secondary cleaning: taking out the chip, placing the chip in a chip cleaning box added with cleaning fluid, placing the chip on a horizontal shaking table, and cleaning; after completion with ddH2O cleaning;
7) and (3) drying: placing the chip in a chip dryer for centrifugal drying;
8) scanning;
9) extracting data;
10) data processing: based on the SNR, the samples were subjected to statistical analysis to determine candidate autoantibody markers.
Further, the rewarming in the above steps is to take the chip out of a refrigerator at-80 ℃, rewarm for half an hour at 4 ℃ and rewarm for 15min at room temperature.
Further, the sealing at room temperature in the above steps is performed at room temperature for 3 hr.
Further, the incubation of the serum sample in the above steps specifically comprises: after the sealing is finished, pouring the sealing liquid completely, then quickly adding a serum incubation liquid prepared in advance, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu l, and the shaking table is laterally swung at 20rpm and incubated overnight at 4 ℃; the samples were pre-frozen in a 4 ℃ chromatography cabinet at a temperature of 1: diluting at 100 proportion.
Further, after the chip clamp is removed in one cleaning in the above steps, the chip is placed in a chip cleaning box with cleaning solution, and the chip is cleaned for 3 times, 10min each time, by a horizontal shaking table at room temperature of 80 rpm.
Further, the secondary antibody incubation in the above step specifically means: the secondary antibody is Fluor conjugated Goatanti-human IgG/donkey anti-human IgM, and is shaken at 40rpm on a side, protected from light and kept at room temperature for 60 min. The secondary antibody is preferably 532nm Fluor conjugated coat resistant anti-human IgG/635nm donkey anti-human IgM.
Further, the secondary cleaning in the above steps is specifically: taking out the chip, placing the chip in a chip cleaning box with cleaning solution, and cleaning for 10min at 80rpm on a horizontal shaker, wherein the upper surface of the chip cannot be touched or scratched; after completion with ddH2O washing for 10min 2 times.
Further, the blocking solution in the step is 10% BSA, 1x PBS solution is added, the volume ratio is preferably 3:7, and the mixture is uniformly mixed and placed on ice; adding 1x PBST solution with the volume ratio of 1:9 into 10% BSA serving as an incubation solution, uniformly mixing, and placing on ice; the cleaning solution was 1x PBST and stored in a refrigerator at 4 ℃.
The invention has the beneficial effects that: the invention provides a diagnosis idea of the autoantibody for the liver cancer, and the autoantibody is amplified by an immune system, is easier to detect and is prior to clinical symptoms, and is suitable for early diagnosis. The 16 potential autoantibody markers are selected and established in a marker combination mode, and the combined use of a plurality of markers is an effective strategy, so that the defect of a single molecule can be avoided, the clinical diagnosis capability can be improved, the current situation of liver cancer diagnosis and curative effect monitoring can be changed, and the autoantibody markers have important clinical value for liver cancer diagnosis.
Drawings
FIG. 1 is a schematic diagram of HCC Focused Arrays detection;
FIG. 2 is a summary of 16 candidate autoantibody markers that can distinguish hepatocellular carcinoma from normal;
FIG. 3 summarizes the results of the t-test (two-tailed).
Detailed Description
The invention is further described below with reference to the accompanying drawings. The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the description of the embodiment is only for illustrating and explaining the present invention and is not for limiting the present invention described in detail in the claims.
Example 1
557 patients with hepatocellular carcinoma and 343 healthy controls were examined using a protein chip containing 100 recombinant proteins focused hepatocellular carcinoma, and 16 candidate autoantibody markers with a certain discrimination ability or combined discrimination ability were finally determined by comparison and analysis of response data.
Reagent required for experiment
1) Sealing liquid: 3ml 10% BSA, added to 7ml 1x PBS solution, mixed, placed on ice.
2) Incubation liquid: 1ml 10% BSA, added to 9ml 1x PBST solution, mixed well and placed on ice.
3) Cleaning solution: 1x PBST, stored in a refrigerator at 4 ℃.
Second, experimental operation steps
1) Rewarming: taking out the chip from a refrigerator at-80 deg.C, re-heating in a refrigerator at 4 deg.C for half an hour, and re-heating at room temperature for 15 min;
2) and (3) sealing: fixing the rewarmed chips in 14blocks, adding sealing liquid into each block, placing on a side swing bed, and sealing at room temperature for 3 hr.
3) Incubation of serum samples: after the blocking was completed, the blocking solution was poured out, and then a serum incubation solution prepared in advance was quickly added, 14 serum samples were incubated for each chip, the loading volume of each serum sample was 200. mu.l, and the shaking table was shaken on the side at 20rpm, and incubated overnight at 4 ℃. (samples were first frozen and thawed in a chromatography cabinet at 4 ℃ and diluted in a ratio of 1: 100)
4) Cleaning: taking out the chip and the chip clamp together, sucking out the sample, then quickly adding the PBST with the same volume, and repeating the steps for a plurality of times to ensure that no cross contamination exists among the serum samples when the chip clamp is detached. After the chip clamp was removed, the chip was placed in a chip washing cassette containing washing solution, and washed on a horizontal shaker at room temperature at 80rpm for 3 times, each time for 10 min.
5) And (3) secondary antibody incubation: the secondary antibody was Fluor conjugated coat goat anti-human IgG (532nm)/donkeyanti-human IgM (635nm), and the chip was transferred to an incubation box containing 3ml of the secondary antibody incubation solution, and the incubation box was shaken on the side at 40rpm, protected from light, and left at room temperature for 60 min.
6) Cleaning: the chip was removed (note that the upper surface of the chip was not touched or scratched), placed in a chip washing cassette to which a washing solution was added, and washed 3 times for 10min each on a horizontal shaker at 80 rpm. After completion, the cells were washed 2 times with ddH2O for 10min each.
7) And (3) drying: the chip is placed in a chip drier for centrifugal drying.
8) Scanning: the operation was performed according to the operating specifications and instructions of the scanner (Luxscan. TM. 10K-A).
9) Data extraction: the corresponding GAL file is opened using GenePix Pro 6.0, the chip image and GAL file are aligned as a whole, the auto-align button is pressed, the data is extracted and the GPR is saved.
Thirdly, data processing step
1) Data definition:
f635 Median: the median value of the foreground value of the signal in the 635nm channel refers to the median value of the intensity of all the pixel points corresponding to each signal point, and is used for representing the signal intensity.
B635 Median: the median value of the background value in the 635nm channel refers to the intensity median value of the pixel points in a certain range of the background around each signal point, and is used for representing the background value.
F532 media: the median value of the foreground value of the signal in the 532nm channel refers to the intensity median value of all the pixel points corresponding to each signal point, and is used for representing the signal intensity.
B532 media: the median value of the background value under the 532nm channel refers to the intensity median value of the pixel points in a certain range of the background around each signal point, and is used for representing the background value.
2) For the extracted data, in order to eliminate the deviation caused by the inconsistency of background values among different samples, the ratio of the foreground value to the background value of each protein, namely F mean/B mean, is calculated, and the SNR (signal to noise ratio), namely the mean value of the F mean/B mean of two repeated proteins, is defined on the basis. Based on the SNR, the samples were statistically analyzed.
The 16 candidate autoantibody markers with certain differentiation between hepatocellular carcinoma and normal human are summarized in FIG. 2. the results of the t-test (two-tailed) are summarized in FIG. 3.
Claims (11)
1. A candidate autoantibody marker for distinguishing hepatocellular carcinoma from a normal human, the candidate autoantibody marker having a distinguishing ability or a combined distinguishing ability, characterized in that: including NPM1, DCAF4L2, TMOD1, ciaapn 1, KDM1A, ANKRD13D, ZNF428, CA12, MAS1, C1QTNF3, ASAH1, MS4A3, WTAP, DAB1, SLC44A3, EGFR.
2. The method of screening candidate autoantibody markers for distinguishing between hepatocellular carcinoma and normal humans according to claim 1, wherein: and detecting the liver cell liver cancer patient and healthy person contrast by adopting a protein chip containing 100 recombinant proteins and focusing on the liver cell liver cancer, and determining the candidate autoantibody marker by responding data comparison and analysis.
3. The method of screening candidate autoantibody markers for distinguishing hepatocellular carcinoma from normal humans according to claim 2, comprising the steps of:
1) rewarming;
2) and (3) sealing: fixing 14blocks of fences on the rewarming chip, adding sealing liquid into each block after the blocks are fixed, placing the blocks on a side swing table, and sealing at room temperature.
3) Incubation of serum samples: after the sealing is finished, pouring the sealing liquid completely, then quickly adding the prepared serum incubation liquid, laterally swinging a shaker, and incubating overnight;
4) primary cleaning: taking out the chip and the chip clamp together, sucking out the sample, then quickly adding PBST with the same volume, and repeating the steps for a plurality of times to ensure that no cross contamination exists among serum samples when the chip clamp is detached; after the chip clamp is removed, the chip is placed in a chip cleaning box with cleaning fluid, and is cleaned by a horizontal shaking table at room temperature;
5) and (3) secondary antibody incubation: transferring the chip to an incubation box added with a secondary antibody incubation solution, laterally swinging a table, keeping out of the sun, and keeping at room temperature;
6) secondary cleaning: taking out the chip, placing the chip in a chip cleaning box added with cleaning fluid, placing the chip on a horizontal shaking table, and cleaning; after completion with ddH2O cleaning;
7) and (3) drying: placing the chip in a chip dryer for centrifugal drying;
8) scanning;
9) extracting data;
10) data processing: based on the SNR, the sample is subjected to statistical analysis to determine the candidate autoantibody markers.
4. The method of claim 3, wherein the rewarming is performed by removing the chip from a-80 ℃ freezer, rewarming the chip in a 4 ℃ freezer for half an hour, and then rewarming the chip at room temperature for 15 min.
5. The method of screening candidate autoantibody markers for distinguishing between hepatocellular carcinoma and normal humans according to claim 3, wherein the blocking is performed at room temperature for 3 hr.
6. The method of claim 3, wherein the incubation of the serum sample comprises: after the sealing is finished, pouring the sealing liquid completely, then quickly adding a serum incubation liquid prepared in advance, wherein each chip can incubate 14 serum samples, the sample loading volume of each serum sample is 200 mu l, and the shaking table is laterally swung at 20rpm and incubated overnight at 4 ℃; the samples were pre-frozen in a 4 ℃ chromatography cabinet at a temperature of 1: diluting at 100 proportion.
7. The method of claim 3, wherein after removing the chip holder in one washing step, the chip is washed 3 times for 10min in a horizontal shaker at room temperature and 80 rpm.
8. The method of claim 3, wherein the secondary antibody is Fluor conjugated coat resistant anti-human IgG/donkey anti-human IgM, shaking table 40rpm, protected from light, and room temperature 60 min.
9. The method of claim 8, wherein the secondary antibody is 532nm Fluor conjugated coat goat anti-human IgG/635nm donkeyanti-human IgM.
10. The method of claim 3, wherein the secondary washing comprises: taking out the chip, placing the chip in a chip cleaning box with cleaning solution, and cleaning for 10min at 80rpm on a horizontal shaker, wherein the upper surface of the chip cannot be touched or scratched; after completion with ddH2O washing for 10min 2 times.
11. The method of claim 3, wherein the blocking solution is 10% BSA, and 1x PBS solution is added at a volume ratio of 3:7, mixed, and placed on ice; adding 1x PBST solution into 10% BSA as an incubation solution in a volume ratio of 1:9, uniformly mixing, and placing on ice; the cleaning solution was 1x PBST and stored in a refrigerator at 4 ℃.
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