CN102279264A - Preparation process for chip with 48 malignant tumor marker antibodies - Google Patents
Preparation process for chip with 48 malignant tumor marker antibodies Download PDFInfo
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- CN102279264A CN102279264A CN2010101950535A CN201010195053A CN102279264A CN 102279264 A CN102279264 A CN 102279264A CN 2010101950535 A CN2010101950535 A CN 2010101950535A CN 201010195053 A CN201010195053 A CN 201010195053A CN 102279264 A CN102279264 A CN 102279264A
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Abstract
The invention specifically provides a preparation process for a chip with 48 malignant tumor marker antibodies, belonging to the technical field of biological engineering. In the invention, the steps of cell fusion, screening of hybridoma cells, specific screening of monoclonal antibodies, purification and preservation of the monoclonal antibodies, marking of anti-human C3, matrix spotting of the monoclonal antibodies, hybridization of chips and the like in the preparation process enable the technical problems of great consumption of time, great consumption of labor, poor repeatability of results and the like in the prior art to be overcome. The chip prepared in the invention can be directly used in research on detection and prevention of a plurality of tumors by medical and scientific research institutions and has a high application value in the field of clinical diagnosis; homogeneity of the monoclonal antibodies and specificity of biological activity enable antigen-antibody reaction results to be convenient for quality control and to be beneficial for standardization and normalization. At present, almost all the overseas medical institutions use the technology of biological chips to some extent for diagnosis and prevention of diseases. Utilization of the technology of biological chips for screening and clinical diagnosis of tumors has the characteristics of saving of time and labor, simpleness and rapidness, and is a developmental trend of modern society.
Description
Technical field
The invention belongs to technical field of bioengineering, be meant a kind of preparation method of 48 kinds of malignant tumour mark antibody chips especially
Background technology
This project is mainly used and is founded antibody chip detection technique platform in malignant tumour associated protein fields such as the cancer of the esophagus, liver cancer, breast cancer, prostate cancer, stomach metastatic carcinomas, 48 kinds of malignant tumour marks of independent research antibody chip detection kit (antibody chip) is by Chinese's independent development fully, use this chip, can be in several ways, multiple channel, stage construction detect tiring and content of malignant tumour mark, detection sensitivity is brought up to the ng level by ug, thereby reach more accurate, effect more fully, but its testing result guiding clinical treatment.48 kinds of malignant tumour mark antibody chip detection kit (antibody chip) have great influence to the development that promotes field, China biological high-technology forward position, the international competitiveness that promotes China's biochip technology, product and market is significant, and the method meets the molecular immunology principle fully.At present external nearly all main drugmaker has all adopted biochip technology to seek drug targets to some extent, the toxicity of inspection medicine or spinoff and carry out drug products quantitatively and qualitative.Carry out large-scale drug screening with chip technology and can omit a large amount of animal experiments, shorten the used time of drug screening, thereby drive the research and development of original new drug.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of 48 kinds of malignant tumour mark antibody chips.
Overall technology design of the present invention is: utilize Fusion of Cells, subclone and enzyme-linked immunosorbent assay to carry out the specificity screening technology, the acquisition height is tired, the anti-human malignant lesion's mark of high specific monoclonal antibody, height, specificity are good because this monoclonal antibody is tired, can directly apply to the point sample of chip, complement C3 antibody and examined product in conjunction with the CY3 mark have been set up complete detection system, detect the method for 48 kinds of malignant tumour marker protein content.This antibody chip has highly sensitive, and advantages such as specificity is good, simple to operate, high flux can be used for clinical and monitoring and Quality Control health check-up serum.
The preparation method of 48 kinds of malignant tumour mark antibody chips, it comprises following processing step:
(1) Fusion of Cells: will have greater activity Sp2/0 myeloma cell respectively with the splenocyte suspension of 48 kinds of sensitization in 1: the 10-100 ratio is mixed, adding polyglycol (U.S. Sigma company product) merges cell each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cellular incubation with HAT selective medium (U.S. sea cloning companies produces);
(2) screening hybridoma: in the time of cellular incubation to the 5-10 to be merged days, draw the culture supernatant that occurs clone cell bunch in the hole of 96 well culture plates and detect antibody content with the enzyme-linked immunosorbent assay method, carry out three subclone screenings through limiting dilution, secretion situation according to antibody filters out the antibody-secreting hole that height is tired, with cell enlarged culture in the hole, carry out antigen specific immune histochemistry in-site detecting then, select height to tire, the cell line of high specific is enlarged culture and frozen again;
(3) monoclonal antibody specificity screening: choosing detects the supernatant of antibody titer greater than 1: 10000 positive hole through enzyme-linked immunosorbent assay, carries out specificity and the screening of tiring with 48 kinds of malignant tumour mark antigens respectively.
(4) monoclonal antibody purification storage: specificity is good and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected when the A280nm, 1.44 (absorbance unit) concentration is 1-10mg/ml.
(5) CY3 labelled antibody: with the fluorescein-labelled complement C3 of CY3 antibody.
(6) 48 kinds of malignant tumour mark monoclonal antibody microarray point samples: 48 points of a chip point, point of each monoclonal antibody, the content 0.01-1ng/ml of each point.
Technological parameter in concrete processing step of the present invention and each step is:
Have highly active Sp2/0 myeloma cell in the step (1) and mix in 1: 10 ratio, add PEG cell is merged each other with the ratio of splenocyte, in the mixed cell suspension of two kinds of cells, the 1st minute dropping 4.5ml nutrient solution; At interval 2 minutes Dropwise 5 ml nutrient solutions add nutrient solution 50ml then, and selecting nutrient culture media with HAT is that 1 cells/well is carried out cellular incubation by 36% hole.
Be when cellular incubation is at the bottom of cover 0%～20% hole in the step (2), draw culture supernatant and detect antibody content with the enzyme-linked immunosorbent assay method, secretion situation according to antibody filters out the antibody-secreting hole that height is tired, with the capable again cloning of cell in the hole, the high secretion of choosing specific cell strain enlarged culture or frozen.
Adopt enzyme-linked immunosorbent assay to detect the supernatant of antibody titer in the step (3), carry out specificity and the screening of tiring with 48 kinds of malignant tumour mark antigens respectively greater than 1: 10000 positive hole.
Specificity is good and tire the nutrient solution sucking-off of clone hole high in the step (4), use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected when the A280nm, 1.44 (absorbance unit) concentration is 1-10mg/ml.
What select for use in the step (5) is the mark of CY3 to complement C3 antibody
Adopt 48 kinds of malignant tumour mark monoclonal antibody microarray point samples in the step (6): 48 points of a chip point, point of each monoclonal antibody, the content 0.01-1ng/ml of each point.
Substantive distinguishing features that the present invention is obtained and significant technical progress are: the 48 kinds of monoclonal antibodies of anti-human malignant lesion's mark that obtained are respectively through AKTA protein purification instrument FPLC purifying, its height of tiring, specificity are very good, can directly the antibody of limiting dilution be put on chip, with the complement C3 antibody of mark with detect sample and hybridize, detect by present advanced person's chip detection instrument.This technology and traditional detection method relatively have fast, the characteristics of simple operation, high throughput testing; And can carry out qualitative detection quickly and accurately.
The method disclosed in the present is that 48 kinds of malignant tumour marks of high-purity are cultivated and thereby a large amount of histocyte original position specificity screenings obtains highly to tire, 48 kinds of anti-human malignant lesion's mark monoclonal antibodies of high specific through Fusion of Cells, cloning, and these antibody can directly apply to clinical detection and fundamental research.Monoclonal antibody has great using value in biology and medical research field, be part important in the affinity chromatography, is antibody main in the SABC, is the novel agent in the immunity inspection, is the guiding weapon of biological therapy.As the detectable of malignant tumour mark, anti-human malignant lesion's mark monoclonal antibody can be given full play to its advantage.Its high specificity of monoclonal antibody can improve the specificity of antigen-antibody reaction greatly, has reduced possible cross reaction, makes the test findings confidence level bigger.The homogeneity of monoclonal antibody and biologically active unicity make the antigen-antibody reaction result be convenient to quality control, are beneficial to standardization and standardization.
Its every index is as follows:
1, highly active Sp2/0 myeloma cell mixes in 1: 10 ratio with the ratio of splenocyte
2, positive colony hole enzyme-linked immunosorbent assay is tired greater than 1: 10000
3,48 points of a chip point, point of each monoclonal antibody, the content 0.01-1ng/ml of each point.
4,48 kinds of malignant tumour mark monoclonal antibodies are as follows: CK18, CK19, Cyclin-D1, CD34, ER, PR, P53, Calcitonin, PSA, P63, CEA, S-100, Vimentin, AR, C-erbB-2, PCNA, Ki-67, HSP70, AE1, AE3, P504S, CK34BE, CA153, CA125, CA199, ECG2, IMP1, Ras, CSK, Erbb2, p90, p16, Calnuc, CyclinE, CDK2, CIAP, RalA, p62, CyclinB1, Koc, CK20, EMA, CA72-4, E-Cadherin, C-KIT, Cathepsin, MDM2, Hepatocyte
Embodiment
Below in conjunction with embodiment the present invention is described further:
The preparation method of 48 kinds of malignant tumour mark antibody chips is characterized in that it comprises following processing step:
(1) Fusion of Cells: will have greater activity Sp2/0 myeloma cell respectively with the splenocyte suspension of 48 kinds of sensitization in 1: the 10-100 ratio is mixed, adding polyglycol (U.S. Sigma company product) merges cell each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cellular incubation with HAT selective medium (U.S. sea cloning companies produces);
(2) screening hybridoma: in the time of cellular incubation to the 5-10 to be merged days, draw the culture supernatant that occurs clone cell bunch in the hole of 96 well culture plates and detect antibody content with the enzyme-linked immunosorbent assay method, carry out three subclone screenings through limiting dilution, secretion situation according to antibody filters out the antibody-secreting hole that height is tired, with cell enlarged culture in the hole, carry out antigen specific immune histochemistry in-site detecting then, select height to tire, the cell line of high specific is enlarged culture and frozen again;
(3) monoclonal antibody specificity screening: choosing detects the supernatant of antibody titer greater than 1: 10000 positive hole through enzyme-linked immunosorbent assay, carries out specificity and the screening of tiring with 48 kinds of malignant tumour mark antigens respectively.
(4) monoclonal antibody purification storage: specificity is good and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected when the A280nm, 1.44 (absorbance unit) concentration is 1-10mg/ml.
(5) CY3 labelled antibody: with the fluorescein-labelled complement C3 of CY3 antibody.
(6) 48 kinds of malignant tumour mark monoclonal antibody microarray point samples: 48 points of a chip point, point of each monoclonal antibody, the content 0.01-1ng/ml of each point.
Adopt the enzyme-linked immunosorbent assay method to measure antiserum after step (1) finishes, this method is made up of following operation steps:
A, bag quilt:
With 48 kinds of dilutions in each 1: 500 of the malignant tumour mark in the step (1), bag is by 96 hole polyethylene boards with the carbonate buffer solution of 50mmol/L, pH=9, and vacuum is drained, and it is standby to seal 4 ℃ of preservations.
B, sealing:
Every hole adds pH and is 7.4 phosphate buffer 200 μ l washing, includes 1% lowlenthal serum;
C, application of sample:
(1: the 5000-10000 dilution), every plate is established a normal control, positive control and blank (phosphate buffer) to 7 days cell culture supernatant 50-100 μ l, washing after every hole adding Fusion of Cells;
D, adding ELIAS secondary antibody every hole 100-200 μ l, washing;
E, colour developing:
Every hole adds substrate 50-100 μ l;
F, colorimetric:
With the blank zeroing, the 405nm wavelength is measured optical density (O.D);
G, result judge: P/N=measures sample O.D average/negative serum O.D average, and P/N 〉=2.1 are positive.
Step (2) is in the time of to be merged cellular incubation to the 5-10 days, draw the culture supernatant that occurs clone cell bunch in the hole of 96 well culture plates and detect antibody content with the enzyme-linked immunosorbent assay method, carry out three subclone screenings through limiting dilution, secretion situation according to antibody filters out the antibody-secreting hole that height is tired, with cell enlarged culture in the hole, carry out antigen specific immune histochemistry in-site detecting then, select height to tire, the cell line of high specific is enlarged culture and frozen again;
Step (3) choosing detects the supernatant of antibody titer greater than 1: 10000 positive hole through enzyme-linked immunosorbent assay, carries out specificity and the screening of tiring with 48 kinds of malignant tumour mark antigens respectively.
Step (4) is good with specificity and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected when the A280nm, 1.44 (absorbance unit) concentration is 1-10mg/ml.
Step (5) the fluorescein-labelled complement C3 of CY3 antibody.
48 points of (6) chip points of step, point of each monoclonal antibody, the content 0.01-1ng/ml of each point.
These 48 kinds of malignant tumour mark antibody chips are expressed all positive through the specificity screening of 30 batches malignant tumour mark antigen, carry out specificity screening with 10 kinds 10 batches normal structure and be expressed as feminine gender.Show that this chip specificity is very high, be fit to offer medical treatment and scientific research institution carries out the evaluation and the Quality Control evaluation of malignant tumour mark, this antibody also can be made into SABC or the enzyme-linked immunosorbent assay kit is carried out basic medical research.
Claims (10)
1.48 plant the preparation method of malignant tumour mark antibody chip, it is characterized in that it comprises following processing step:
(1) Fusion of Cells: will have greater activity Sp2/0 myeloma cell respectively with the splenocyte suspension of 48 kinds of sensitization in 1: the 10-100 ratio is mixed, adding polyglycol (U.S. Sigma company product) merges cell each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cellular incubation with HAT selective medium (U.S. sea cloning companies produces);
(2) screening hybridoma: in the time of cellular incubation to the 5-10 to be merged days, draw the culture supernatant that occurs clone cell bunch in the hole of 96 well culture plates and detect antibody content with the enzyme-linked immunosorbent assay method, carry out three subclone screenings through limiting dilution, secretion situation according to antibody filters out the antibody-secreting hole that height is tired, with cell enlarged culture in the hole, carry out antigen specific immune histochemistry in-site detecting then, select height to tire, the cell line of high specific is enlarged culture and frozen again;
(3) monoclonal antibody specificity screening: choosing detects the supernatant of antibody titer greater than 1: 10000 positive hole through enzyme-linked immunosorbent assay, carries out specificity and the screening of tiring with 48 kinds of malignant tumour mark antigens respectively.
(4) monoclonal antibody purification storage: specificity is good and tire the nutrient solution sucking-off of clone hole high, use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected when the A280nm, 1.44 (absorbance unit) concentration is 1-10mg/ml.
(5) CY3 labelled antibody: with the fluorescein-labelled complement C3 of CY3 antibody.
(6) 48 kinds of malignant tumour mark monoclonal antibody microarray point samples: 48 points of a chip point, point of each monoclonal antibody, the content 0.01-1ng/ml of each point.
2. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1, it is characterized in that described step (1) will have greater activity Sp2/0 myeloma cell respectively with the splenocyte suspension of 48 kinds of sensitization in 1: the 10-100 ratio is mixed, adding polyglycol (U.S. Sigma company product) merges cell each other, in the mixed cell suspension of two kinds of cells, drip nutrient solution, carry out cellular incubation with HAT selective medium (U.S. sea cloning companies produces); (6) malignant tumour mark monoclonal antibody microarray point sample: 48 points of a chip point, detect the range of linearity 0.01～10ng/ml.
3. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1 and 2 is characterized in that adopting the enzyme-linked immunosorbent assay method to measure antiserum after described step (1) finishes, and this method is made up of following operation steps:
A, bag quilt:
Carbonate buffer solution with 50mmol/L, pH=9 diluted 48 kinds of malignant tumour marks in the step (1) in each 1: 500, and bag is by 96 hole polyethylene boards, and vacuum is drained, and it is standby to seal 4 ℃ of preservations.
B, sealing:
Every hole adds pH and is 7.4 phosphate buffer 200 μ l washing, includes 1% lowlenthal serum;
C, application of sample:
(1: the 5000-10000 dilution), every plate is established a normal control, positive control and blank (phosphate buffer) to 7 days cell culture supernatant 50-100 μ l, washing after every hole adding Fusion of Cells;
D, adding ELIAS secondary antibody every hole 100-200 μ l, washing;
E, colour developing:
Every hole adds substrate 50-100 μ l;
F, colorimetric:
With the blank zeroing, the 405nm wavelength is measured optical density (O.D);
G, result judge: P/N=measures sample O.D average/negative serum O.D average, and P/N 〉=2.1 are positive.
4. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 3, it is characterized in that the process conditions among the described step c are that every hole adds 1: 7 days cell culture supernatant 50-100 μ l after the Fusion of Cells after the 5000-10000 dilution, every plate is established a normal control, positive control and blank (phosphate buffer), temperature is that 37 ℃, time are 1 hour, washs 3 times.
5. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1, the ratio that it is characterized in that having in the described step (1) highly active Sp2/0 myeloma cell and splenocyte is in 1: the ratio of 10-100 is mixed, 30-60 adds 45%PEG (molecular weight: 4000) gradually in second, static 90-120 second, cell is merged each other, in the mixed cell suspension of two kinds of cells, the 1st minute dropping 4.5ml1640 nutrient solution; At interval 2 minutes Dropwise 5 ml1640 nutrient solutions add RPMI-1640 then to 50ml, and 1500rpm/ minute centrifugal 10 minutes is that 1 cells/well is carried out cellular incubation with HAT selective medium (U.S. sea cloning companies produces) by the hole of 20-50%.
6. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1, it is characterized in that in the described step (2) it being when cellular incubation is at the bottom of cover 0%～20% hole, draw culture supernatant and detect antibody content with the enzyme-linked immunosorbent assay method, secretion situation according to antibody filters out the antibody-secreting hole that height is tired, with the capable again cloning of cell in the hole, carry out the immunohistochemistry of antigen-specific then and measure, the high secretion of choosing specific cell strain enlarged culture or frozen.
7. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1 is characterized in that carrying out specificity and the screening of tiring with 48 kinds of malignant tumour mark antigens respectively in the described step (3).
8. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1, it is good with specificity and tire the nutrient solution sucking-off of clone hole high to it is characterized in that in the described step (4), use AKTA-FPLC protein purification instrument (manufacturing of AM General company) that IgG monoclonal antibody culture supernatant solution is collected when the A280nm, 1.44 (absorbance unit) concentration is 1-10mg/ml.
9. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1 is characterized in that described step (5) carries out the CY3 mark to the antibody of its C3 complement.
10. the preparation method of 48 kinds of malignant tumour mark antibody chips according to claim 1, it is characterized in that (6) 48 kinds of malignant tumour mark monoclonal antibodies of described step microarray point sample: 48 points of a chip point, sensing range 0.01～10ng/ml.
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Cited By (7)
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| CN103197071A (en) * | 2013-03-22 | 2013-07-10 | 广州兹尼生物科技有限公司 | Preparation and application of human apoptosis factor quantitative antibody chip |
| CN103869068A (en) * | 2012-12-18 | 2014-06-18 | 广州瑞博奥生物科技有限公司 | Antibody chip kit for diagnosis of various tumors |
| CN104062431A (en) * | 2014-07-01 | 2014-09-24 | 上海理工大学 | Method for screening monoclonal antibody hybridoma by utilization of visual antigen chip |
| CN105891496A (en) * | 2014-12-09 | 2016-08-24 | 上海华盈生物医药科技有限公司 | Tyrosine kinase inhibitor targeted medication guidance antibody chip and detection method |
| CN105917230A (en) * | 2013-11-11 | 2016-08-31 | 伊缪诺维亚公司 | Method, array and use thereof for determining pancreatic cancer |
| US11320436B2 (en) | 2020-07-16 | 2022-05-03 | Immunovia Ab | Methods, arrays and uses thereof |
| US11525832B2 (en) | 2007-03-27 | 2022-12-13 | Immunovia Ab | Protein signature/markers for the detection of adenocarcinoma |
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| US11525832B2 (en) | 2007-03-27 | 2022-12-13 | Immunovia Ab | Protein signature/markers for the detection of adenocarcinoma |
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| CN105917230A (en) * | 2013-11-11 | 2016-08-31 | 伊缪诺维亚公司 | Method, array and use thereof for determining pancreatic cancer |
| CN104062431A (en) * | 2014-07-01 | 2014-09-24 | 上海理工大学 | Method for screening monoclonal antibody hybridoma by utilization of visual antigen chip |
| CN105891496A (en) * | 2014-12-09 | 2016-08-24 | 上海华盈生物医药科技有限公司 | Tyrosine kinase inhibitor targeted medication guidance antibody chip and detection method |
| US11320436B2 (en) | 2020-07-16 | 2022-05-03 | Immunovia Ab | Methods, arrays and uses thereof |
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