CN1044384C - Building of anti-type-C hepatitis virus antigen monoclonal antibody cell strain and monoclonal antibody thereof - Google Patents
Building of anti-type-C hepatitis virus antigen monoclonal antibody cell strain and monoclonal antibody thereof Download PDFInfo
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- CN1044384C CN1044384C CN94115017A CN94115017A CN1044384C CN 1044384 C CN1044384 C CN 1044384C CN 94115017 A CN94115017 A CN 94115017A CN 94115017 A CN94115017 A CN 94115017A CN 1044384 C CN1044384 C CN 1044384C
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Abstract
The present invention establishes five hybridoma cell strains capable of secreting specific monoclonal antibodies which can be respectively combined with antigens of a nuclear zone, a membranous zone, an NS3 zone, an NS4 zone and an NS5 zone of a non-A, non-B hepatitis virus, and the monoclonal antibodies secreted by the hybridoma cell strains can be obtained. A high-sensitivity specific antigen-antibody detecting method can be established by using the anti-HCV specific monoclonal antibodies, and HCV virus antigens can be separated and identified.
Description
Hepatitis C is the Pandemic infection disease, and is main by blood transfusion and blood products propagation.Cloned and given expression to the protein (C-100-3) of the non-structural area 3～4 of coding hepatitis C virus HCV (NS3～4) partial sequence in 1988 by American scholar first and set up the immunological method of artificial antigen in succession, very important effect has been played in the control that HCV propagates for the mensuration antibody on basis.But because HCV virus separation difficulty is heavy, virological investigation does not obtain breakthrough as yet, thus the unique indication that all infects as HCV with HCV antigen/antibody combination at present.But used antigen is the antigen of artificial synthetic polypeptide or gene engineering expression, no matter is still certainly existing certain difference in primary structure on sterie configuration with native antigen.Strengthen the further investigation of HCV virus thus and set up high responsive antigen-antibody measuring method that very important meaning is arranged.Can make up the fundamental research of special antigen-antibody measuring method, the separation that is used for the HCV virus antigen, evaluation and virus and virus infection of high sensitivity with the specific monoclonal antibody of anti-HCV.Be not seen in report both at home and abroad as yet with the specific mouse monoclonal antibody of HCV.
The objective of the invention is to prepare the specific mouse monoclonal antibody of anti-HCV, we have successfully prepared core area, film district, the antigenic monoclonal antibody specific of NS3, NS4 and NS5 district of anti-HCV with the not synantigen of HCV genes encoding, thereby lay the foundation for the fundamental research of HCV and application.
One, the foundation of cell strain of monoclonal antibody
(1), the foundation of anti-HCV core area cell strain of monoclonal antibody:
1, immunogenic preparation:
Synthetic core area polypeptide [available from the artificial synthetic polypeptide (20 peptide) of U.S. UBI Bioisystech Co., Ltd] 2mg, BSA4mg/ml PH7.2,0.1M ammonium acetate buffer, drip 0.02M glutaraldehyde 0.5ml, stirring at room 2 hours, change liquid once in 4 ℃ of 0.02M PH7.2 PBS dialysis 48 hours (12 hours time), packing ,-20 ℃ standby.
2, immune animal:
Press polypeptide 20 a μ g/ BALB/c mouse (4-6 age in week), after 0.25ml adds 0.25ml Freund's complete adjuvant (FCA) emulsification, four soles and subcutaneous multi-point injection.Add the incomplete left agent emulsification pneumoretroperitoneum of Fu Shi with doses of antigen after 4 weeks and strengthen, week back eyeball of mouse bloodletting.
3, immunizing potency is measured:
Preparing the 0.01M PH9.6 carbonic acid buffer 96 hole polystyrene plates of 5 μ g/ml polypeptide, spends the night for 4 ℃ in 37 ℃ in 0.1ml/ hole 2 hours.Inferior order, 10% bovine serum, 1% skim-milk 0.02MPH7.2 PBS 0.15ml/ hole, 37 ℃, 2 hours, 4 ℃ were spent the night, and were used for the mouse serum titer and measured.
Mouse serum with 10% bovine serum PBS (the same) with 10
2～10
6Doubly dilution, add 96 orifice plates, 0.1ml/ 37 ℃ in hole, 2 hours, add 2 after washing six times, the anti-mouse of rabbit (Sigma) of the horseradish peroxidase mark of 000 times of dilution after 37 ℃ of 2 hours the same washing, adds 50ul and contains 0.1% (W/V) O-Phenylene Diamine 0.1% (V/V) hydrogen peroxide PH5.0 citric acid phosphoric acid damping fluid.0.1ml/ the hole, room temperature lucifuge 20 minutes is surveyed 492 absorption values, and mouse serum is made negative control before exempting from, and judges tiring of immune mouse serum so that the measured value and ratio 〉=2.1 of control value are positive.
4, cytogamy is built strain:
After last was strengthened, the aseptic mouse spleen of getting was made splenocyte suspension and is mixed by 10: 1 with murine myeloma cell, with molecular weight 6,000 PEG adds 0.7ml as fusogen down at 37 ℃, makes its fusion, select culture medium culturing with HAT, hybridoma can be grown, treat that cell grows to 10
5During/ml concentration, the same immunizing potency assay method screening detects the situation of emiocytosis antibody; Clone with limiting dilution assay on 96 well culture plates with 1 cells/well in secretory antibody positive cell hole, screens positive Kong Yi and go up the continuous clone of method three times, after the enlarged culturing, prepares 10 with the nutrient solution of 10%DMSO
6/ ml cell, the cytogamy 6 strain stably excreting antibody cell strains of once building together.Name JDSHCC39 (CCTCC NO:C94005) respectively, JDSHCC58, JDSHCC81, JDSHCC105, JDSHCC123, JDSHCC221 clone.
(2), the foundation of anti-HCV film region monoclonal antibody cell strain:
1, immunogenic preparation:
To synthesize polypeptide (available from the artificial synthetic polypeptide of U.S. UBI Bioisystech Co., Ltd) 2mg, BSA4mg/ml, the PH7.20.1M ammonium acetate buffer, drip 0.02M glutaraldehyde 0.5ml, stirring at room 2 hours, at 0.02M PH7.2,4 ℃ of dialysis of PBS 48 hours (12 hours) are changed liquid once, packing, and-20 ℃ are standby.
2, immune animal:
Press polypeptide 20 a μ g/ BALB/c mouse (4-6 age in week), after 0.25ml adds the complete left agent of 0.25ml Fu Shi (FCA) emulsification, four soles and subcutaneous multi-point injection.Add freund 's incomplete adjuvant emulsification pneumoretroperitoneum with doses of antigen after 4 weeks and strengthen, week back eyeball of mouse bloodletting.
3, immunizing potency is measured:
Prepare the 0.01M PH9.6 carbonic acid buffer 96 hole polystyrene plates of 5 μ g/ml polypeptide, next day is spent the night for 4 ℃ in 37 ℃ in 0.1ml/ hole 2 hours, 10% bovine serum, 1% skim-milk 0) .02MPH7.2 PBS 0.15ml/ hole, 37 ℃, 2 hours, 4 ℃ are spent the night, and are used for the mouse serum titer and measure.
Mouse serum with 10% bovine serum PBS (the same) with 10
2～10
6Doubly dilution, add 96 orifice plates, 37 ℃ in 0.1ml/ hole, 2 hours, add 2 after washing six times, the anti-mouse of rabbit (Sigma) of the horseradish peroxidase mark of 000 times of dilution, after 37 ℃ of 2 hours the same washing, every hole adds 100ul substrate solution (0.1% (W/V) O-Phenylene Diamine 0.1% (V/V) hydrogen peroxide PH5.0 citric acid phosphoric acid damping fluid), room temperature lucifuge 20 minutes, survey the 492nm absorption value, the ashamed negative control of mouse serum before exempting from is judged tiring of immune mouse serum so that the measured value and ratio 〉=2.1 of control value are positive.
4, cytogamy is built strain:
After last was strengthened, the aseptic mouse spleen of getting was made splenocyte suspension and is mixed by 10: 1 with murine myeloma cell, with molecular weight 6,000 PEG adds 0.7ml as fusogen down at 37 ℃, makes its fusion, select culture medium culturing with HAT, hybridoma can be grown, treat that cell grows to 10
5During/ml concentration, the same immunizing potency assay method screening detects the situation of emiocytosis antibody; Clone with limiting dilution assay on 96 well culture plates with 1 cells/well in secretory antibody positive cell hole, and screen positive Kong Yi and go up the continuous clone of method three times, after the enlarged culturing, the nutrient solution of 10%DMSO preparation cell to 10
6/ ml liquid nitrogen cryopreservation.The cytogamy 4 strain stably excreting antibody cell strains of once building together.Difference called after JDSHCE-44, JDSHCE-56, JDSHCE-125 (CCTCC NO:C94009), JDSHCE-246 clone.
(3), anti-HCVNS3 region monoclonal antibody cell strain is set up:
1. immune animal: press antigen [available from Japan's eastern combustion company gene engineering antigen (MW:20KD)] 20 a μ g/ BALB/c mouse (4-6 age in week), after 0.25ml adds 0.25ml Freund's complete adjuvant (FCA) emulsification, four soles and subcutaneous multi-point injection.Add Freund's incomplete adjuvant emulsification pneumoretroperitoneum with doses of antigen after 4 weeks and strengthen, week back eyeball of mouse bloodletting.
2, immunizing potency is measured:
The 0.01M PH9.6 carbonic acid buffer 96 hole polystyrene plates of preparation 5ug/ml polypeptide, next day is spent the night for 4 ℃ in 37 ℃ in 0.1ml/ hole 2 hours, 10% bovine serum, 1% skim-milk 0.02MPH7.2 PBS 0.15ml/ hole, 37 ℃, 2 hours, 4 ℃ are spent the night, and are used for the mouse serum titer and measure.
Mouse serum with 10% bovine serum PBS (the same) with 10
2～10
6Doubly dilution, add 96 orifice plates, 0.1ml/ 37 ℃ in hole 2 hours, adds 2 after washing six times, the anti-mouse of rabbit (Sigma) of the horseradish peroxidase mark of 000 times of dilution, after 37 ℃ of 2 hours the same washing, add 50ul and contain 0.1% (W/V) O-Phenylene Diamine 0.1% (V/V) hydrogen peroxide PH5.0 citric acid phosphoric acid damping fluid, 0.1ml/ hole, room temperature lucifuge 20 minutes, OD
492Measure absorption value, mouse serum is made negative control before exempting from, and judges tiring of immune mouse serum so that the measured value and ratio 〉=2.1 of control value are positive.
3, cytogamy is built strain:
The aseptic spleen of getting of the strengthened mouse of last is made splenocyte suspension and is mixed by 10: 1 with murine myeloma cell, with molecular weight 6,000 PEG adds 0.7ml as fusogen down at 37 ℃, makes its fusion, select culture medium culturing with AHT, hybridoma can be grown, treat that cell grows to 10
5During/ml concentration, the same immunizing potency assay method screening detects the situation of emiocytosis antibody; Clone with limiting dilution assay on 96 well culture plates with 1 cells/well in secretory antibody positive cell hole, and screen positive Kong Yi and go up the continuous clone of method three times, after the enlarged culturing, the nutrient solution of 10%DMSO preparation cell to 10
6/ ml liquid nitrogen cryopreservation.The cytogamy 5 strain stably excreting antibody cell strains of once building together.Name JDSHCNS3-1 (CCTCC NO:C94006) respectively, JDSHCNS3-3, JDSHCNS3-6, JDSHCNS3-8, JDSHCNS3-9.
(4), anti-HCVNS4 region monoclonal antibody cell strain is set up:
1, immunogenic preparation:
Synthetic polypeptide [available from the artificial synthetic polypeptide (25 peptide) of Canadian Yes company] 2mg, BSA4mg/ml PH7.20.1M ammonium acetate buffer, drip 0.02M glutaraldehyde 0.5ml, stirring at room 2 hours, 0.02M PH7.2 PBS4 ℃ of dialysis 48 hours (12 hours) changed liquid once, packing ,-20 ℃ standby.
2, immune animal:
Press a polypeptide 20ug/ BALB/c mouse (4-6 age in week), after 0.25ml adds 0.25ml Fu Shi Freund's complete adjuvant (FCA) emulsification, four soles and subcutaneous multi-point injection.Add freund 's incomplete adjuvant emulsification pneumoretroperitoneum with doses of antigen after 4 weeks and strengthen, week back eyeball of mouse bloodletting.
3, immunizing potency is measured:
The 0.01M PH9.6 carbonic acid buffer 96 hole polystyrene plates of preparation 5ug/ml polypeptide, next day is spent the night for 4 ℃ in 37 ℃ in 0.1ml/ hole 2 hours, 10% bovine serum, 1% skim-milk 0.02MPH7.2 PBS 0.15ml/ hole, 37 ℃, 2 hours, 4 ℃ are spent the night, and are used for the mouse serum titer and measure.
Mouse serum with 10% bovine serum PBS (the same) with 102～106 times of dilutions, add 96 orifice plates, 0.1ml/ 37 ℃ in hole 2 hours, adds 2 after washing six times, the anti-mouse of rabbit (Sigma) of the horseradish peroxidase mark of 000 times of dilution, after 37 ℃ of 2 hours the same washing, add 50ul and contain 0.1% (W/V) O-Phenylene Diamine 0.1% (V/V) hydrogen peroxide PH5.0 citric acid phosphoric acid damping fluid, 0.1ml/ hole, room temperature lucifuge 20 minutes, OD
492Measure absorption value, the ashamed negative control of mouse serum before exempting from is judged tiring of immune mouse serum so that the measured value and ratio 〉=2.1 of control value are positive.
4, cytogamy is built strain:
After last was strengthened, the aseptic mouse spleen of getting was made splenocyte suspension and is mixed by 10: 1 with murine myeloma cell, with molecular weight 6,000 PEG adds 0.7ml as fusogen down at 37 ℃, makes its fusion, select culture medium culturing with HAT, hybridoma can be grown, treat that cell grows to 10
5During/ml concentration, the same immunizing potency assay method screening detects the situation of emiocytosis antibody; Clone with limiting dilution assay on 96 well culture plates with 1 cells/well in secretory antibody positive cell hole, and screen positive Kong Yi and go up the continuous clone of method three times, after the enlarged culturing, the nutrient solution of 10%DMSO preparation cell to 10
6/ ml liquid nitrogen cryopreservation.The cytogamy 3 strain stably excreting antibody cell strains of once building together.Difference called after JDSHCNS4-93 (CCTCC NO:C94007), JDSHCNS4-40, JDSHCNS4-13.
(5) anti-HCVNS5 region monoclonal antibody cell strain is set up:
1, immunogenic preparation:
Synthetic polypeptide [available from the artificial synthetic polypeptide (20 peptide) of U.S. UB I Bioisystech Co., Ltd] 2mg, BSA4mg/mlPH7.2 0.1M ammonium acetate buffer, drip 0.02M glutaraldehyde 0.5ml, stirring at room 2 hours, 0.02M PH7.2 PBS4 ℃ of dialysis 48 hours (12 hours) changed liquid once, packing ,-20 ℃ standby.
2, immune animal:
Press a polypeptide 20ng/ BALB/c mouse (4-6 age in week), after 0.25ml adds 0.25ml Freund's complete adjuvant (FCA) emulsification, four soles and subcutaneous multi-point injection.Add Freund's incomplete adjuvant emulsification pneumoretroperitoneum with doses of antigen after 4 weeks and strengthen, week back eyeball of mouse bloodletting.
3, immunizing potency is measured:
The 0.01M PH9.6 carbonic acid buffer 96 hole polystyrene plates of preparation 5ug/ml polypeptide, next day is spent the night for 4 ℃ in 37 ℃ in 0.1ml/ hole 2 hours, 10% bovine serum, 1% skim-milk 0.02M PH7.2 PBS 0.15ml/ hole, 37 ℃, 2 hours, 4 ℃ are spent the night, and are used for the mouse serum titer and measure.
Mouse serum with 10% bovine serum PBS (the same) with 10
2～10
6Doubly dilution adds 96 orifice plates, 37 ℃ in 0.1ml/ hole, 2 hours, wash the anti-mouse of rabbit (Sigma) that adds the horseradish peroxidase mark of 2,000 times of dilutions after six times, after 37 ℃ of 2 hours the same washing, add 50ul and contain 0.1% (W/V) O-Phenylene Diamine 0.1% (V/V) hydrogen peroxide PH5.0 citric acid phosphoric acid damping fluid, 0.1ml/ the hole, room temperature lucifuge 20 minutes is surveyed the 492nm absorption value, mouse serum is made negative control before exempting from, and judges tiring of immune mouse serum so that the measured value and ratio 〉=2.1 of control value are positive.
4, cytogamy is built strain:
After last was strengthened, the aseptic mouse spleen of getting was made splenocyte suspension and is mixed by 10: 1 with murine myeloma cell, with molecular weight 6,000 PEG adds 0.7ml as fusogen down at 37 ℃, makes its fusion, select culture medium culturing with HAT, hybridoma can be grown, treat that cell grows to 10
5During/ml concentration, the same immunizing potency assay method screening detects the situation of emiocytosis antibody; Clone with limiting dilution assay on 96 well culture plates with 1 cells/well in secretory antibody positive cell hole, screens positive Kong Yi and go up the continuous clone of method three times, and after the enlarged culturing, the nutrient solution of 10%DMSO preparation cell is to the 106/ml liquid nitrogen cryopreservation.The cytogamy 2 strain stably excreting antibody cell strains of once building together.Difference called after JDSHCNS5-1 (CCTCC NO:C94008), JDSHCNS5-2.Two, the grand antibody of MONOCLONAL ANTIBODIES SPECIFIC FOR and the anti-HCV core area of HRP mark () Dan Kekang: 1, MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying:
Collect the culturing cell counting and be made into 5 * 10
6The concentration abdominal injection of/ml, every 0.5ml BALB/c mouse while injecting fluid paraffin 0.5ml/ only gathers ascites 3,000 and left the heart 30 minutes after 7～10 days, and getting supernatant is odd contradictive hydroperitoneum; Add the PH4.0 0.06M acetate buffer of 2 times of volumes with the ascites of 1 times of volume, drip n-caprylic acid by 3.3% (V/V), stirring at room 20 minutes, 10,000 left the heart 20 minutes, and supernatant filters 4 ℃ of dialysed overnight of the neutral PBS in back; Next day, the concentration by 25.8% added required solid ammonium sulfate 4 ℃ 〉=2 hours, 3,000 left the heart 20 minutes, precipitation adds the saturated ammonium sulphate of 1/2 volume with the center P BS dissolving of 1 times of volume, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, precipitation is with the neutral PBS dissolving of 1/2 volume, with 48 hours (changing liquid once in 12 hours) of 4 ℃ of dialysis of liquid.The Lowry method is surveyed packing-20 ℃ preservation after the proteic concentration.2, HRP-monoclonal antibody binding substances preparation:
4mg HRP is dissolved in the 1ml water, add 0.1ml 0.1M sodium periodate oxidation 20 minutes, neutral PBS4 ℃ of dialysed overnight, add next day to add behind the 25ul 0.2M yellow soda ash and use PH9.60.01M carbonic acid buffer equilibrated 10mg monoclonal antibody in advance, the room temperature lucifuge stirred 2 hours, the sodium borohydride that adds 0.1ml4mg/ml, 4 ℃ 2 hours, 4 ℃ of dialysed overnight of neutral PBS add isopyknic saturated ammonium sulphate next day, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, after precipitation is washed twice with 50% ammonium sulfate, the neutral PBS dissolving of 1ml ,-20 ℃ standby.(2) anti-HCV film region monoclonal antibody: 1, Monoclonal Antibody and purifying:
Collect the culturing cell counting and be made into 5 * 10
6The concentration abdominal injection of/ml, every 0.5ml BALB/c mouse while injecting fluid paraffin 0.5ml/ only gathers ascites 3,000 and left the heart 30 minutes after 7～10 days, and getting supernatant is odd contradictive hydroperitoneum; Add the PH4.0 0.06M acetate buffer of 2 times of volumes with the ascites of 1 times of volume, drip n-caprylic acid by 3.3% (V/V), stirring at room 20 minutes, 10,000 left the heart 20 minutes, and supernatant filters 4 ℃ of dialysed overnight of the neutral PBS in back; Next day, the concentration by 25.8% added the required solid ammonium sulfate of people 4 ℃ 〉=2 hours, 3,000 left the heart 20 minutes, precipitation adds the saturated ammonium sulphate of 1/2 volume with the neutral PBS dissolving of 1 times of volume, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, precipitation is with the center P BS dissolving of 1/2 volume, with 48 hours (changing liquid once in 12 hours) of 4 ℃ of dialysis of liquid.The Lowry method is surveyed packing-20 ℃ preservation after the proteic concentration.2, HRP-monoclonal antibody binding substances preparation:
4mg HRP is dissolved in the 1ml water, add 0.1ml 0.1M sodium periodate oxidation 20 minutes, neutral PBS4 ℃ of dialysed overnight, add next day to add behind the 25ul 0.2M yellow soda ash and use PH9.60.01M carbonic acid buffer equilibrated 10mg monoclonal antibody in advance, the room temperature lucifuge stirred 2 hours, the sodium borohydride that adds 0.1ml4mg/ml, 4 ℃ 2 hours, 4 ℃ of dialysed overnight of neutral PBS add isopyknic saturated ammonium sulphate next day, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, after precipitation is washed twice with 50% ammonium sulfate, the neutral PBS dissolving of 1ml ,-20 ℃ standby.(3) anti-HCVNS3 region monoclonal antibody: 1, Monoclonal Antibody and purifying:
Collect the culturing cell counting and be made into 5 * 10
6The concentration abdominal injection of/ml, every 0.5ml BALB/c mouse while injecting fluid paraffin 0.5ml/ only gathers ascites 3,000 and left the heart 30 minutes after 7～10 days, and getting supernatant is odd contradictive hydroperitoneum; Add the PH4.0 0.06M acetate buffer of 2 times of volumes with the ascites of 1 times of volume, drip n-caprylic acid by 3.3% (V/V), stirring at room 20 minutes, 10,000 left the heart 20 minutes, and supernatant filters 4 ℃ of dialysed overnight of the neutral PBS in back; Next day, the concentration by 25.8% added required solid ammonium sulfate 4 ℃ 〉=2 hours, 3,000 left the heart 20 minutes, precipitation adds the saturated ammonium sulphate of 1/2 volume with the center P BS dissolving of 1 times of volume, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, precipitation is with the neutral PBS dissolving of 1/2 volume, with 48 hours (changing liquid once in 12 hours) of 4 ℃ of dialysis of liquid.The Lowry method is surveyed packing-20 ℃ preservation after the proteic concentration.2, HRP one monoclonal antibody binding substances preparation:
4mg HRP is dissolved in the 1ml water, add 0.1ml 0.1M sodium periodate oxidation 20 minutes, neutral PBS4 ℃ of dialysed overnight, add next day to add behind the 25ul 0.2M yellow soda ash and use PH9.60.01M carbonic acid buffer equilibrated 10mg monoclonal antibody in advance, the room temperature lucifuge stirred 2 hours, the sodium borohydride that adds 0.1ml4mg/ml, 4 ℃ 2 hours, 4 ℃ of dialysed overnight of neutral PBS add isopyknic saturated ammonium sulphate next day, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, after precipitation is washed twice with 50% ammonium sulfate, the neutral PBS dissolving of 1ml ,-20 ℃ standby.(4) anti-HCVNS4 region monoclonal antibody: 1, Monoclonal Antibody and purifying:
Collect the culturing cell counting and be made into 5 * 10
6The concentration abdominal injection of/ml, every 0.5ml BALB/c mouse while injecting fluid paraffin 0.5ml/ only gathers ascites 3,000 and left the heart 30 minutes after 7～10 days, and getting supernatant is odd contradictive hydroperitoneum; Add the PH4.0 0.06M acetate buffer of 2 times of volumes with the ascites of 1 times of volume, drip n-caprylic acid by 3.3% (V/V), stirring at room 20 minutes, 10,000 left the heart 20 minutes, and supernatant filters 4 ℃ of dialysed overnight of the neutral PBS in back; Next day, the concentration by 25.8% added required solid ammonium sulfate 4 ℃ 〉=2 hours, 3,000 left the heart 20 minutes, precipitation adds the saturated ammonium sulphate of 1/2 volume with the neutral PBS dissolving of 1 times of volume, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, precipitation is with the neutral PBS dissolving of 1/2 volume, with 48 hours (changing liquid once in 12 hours) of 4 ℃ of dialysis of liquid.The Lowry method is surveyed packing-20 ℃ preservation after the proteic concentration.2, HRP-monoclonal antibody binding substances preparation:
4mg HRP is dissolved in the 1ml water, add 0.1ml 0.1M sodium periodate oxidation 20 minutes, neutral PBS4 ℃ of dialysed overnight, add next day to add behind the 25ul 0.2M yellow soda ash and use PH9.60.01M carbonic acid buffer equilibrated 10mg monoclonal antibody in advance, the room temperature lucifuge stirred 2 hours, the sodium borohydride that adds 0.1ml4mg/ml, 4 ℃ 2 hours, 4 ℃ of dialysed overnight of neutral PBS add isopyknic saturated ammonium sulphate next day, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, after precipitation is washed twice with 50% ammonium sulfate, the neutral PBS dissolving of 1ml ,-20 ℃ standby.(5) anti-HCVNS5 region monoclonal antibody: 1, Monoclonal Antibody and purifying:
Collect the culturing cell counting and be made into 5 * 10
6The concentration abdominal injection of/ml, every 0.5ml BALB/c mouse while injecting fluid paraffin 0.5ml/ only gathers ascites 3,000 and left the heart 30 minutes after 7～10 days, and getting supernatant is odd contradictive hydroperitoneum; Add the PH4.0 0.06M acetate buffer of 2 times of volumes with the ascites of 1 times of volume, drip n-caprylic acid by 3.3% (V/V), stirring at room 20 minutes, 10,000 left the heart 20 minutes, and supernatant filters 4 ℃ of dialysed overnight of the neutral PBS in back; Next day, the concentration by 25.8% added required solid ammonium sulfate 4 ℃ 〉=2 hours, 3,000 left the heart 20 minutes, precipitation adds the saturated ammonium sulphate of 1/2 volume with the center P BS dissolving of 1 times of volume, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, precipitation is with the neutral PBS dissolving of 1/2 volume, with 48 hours (changing liquid once in 12 hours) of 4 ℃ of dialysis of liquid.The Lowry method is surveyed packing-20 ℃ preservation after the proteic concentration.2, HRP-monoclonal antibody binding substances preparation:
4mg HRP is dissolved in the 1ml water, add 0.1ml 0.1M sodium periodate oxidation 20 minutes, neutral PBS4 ℃ of dialysed overnight, add next day to add behind the 25ul 0.2M yellow soda ash and use PH9.60.01M carbonic acid buffer equilibrated 10mg monoclonal antibody in advance, the room temperature lucifuge stirred 2 hours, the sodium borohydride that adds 0.1ml4mg/ml, 4 ℃ 2 hours, 4 ℃ of dialysed overnight of neutral PBS add isopyknic saturated ammonium sulphate next day, 4 ℃ 2 hours, 3,000 left the heart 30 minutes, after precipitation is washed twice with 50% ammonium sulfate, the neutral PBS dissolving of 1ml ,-20 ℃ standby.Three, the anti-HCV core area of immunology character analysis () monoclonal antibody of monoclonal antibody: 1, specific assay:
Measure with the same immunizing potency assay method of the antigen of different zones.Found that outside the core area antigen-reactive, not have specific reaction with other regional antigen of HCV and contrast antigen.2, the monoclonal antibody recognition site is measured:
5 μ g/ml antigen 0.1ml/ holes add 96 orifice plates, 37 ℃ of solid phases are after 2 hours, the monoclonal antibody 50 μ l that add odd contradictive hydroperitoneum 50 μ l and HRP mark, 37 ℃ after 1 hour, add substrate solution. survey the 492nm absorption value, with odd contradictive hydroperitoneum the monoclonal antibody of same HRP mark being suppressed is 100%, with known irrelevant odd contradictive hydroperitoneum to the negative contrast of the inhibition of mark monoclonal antibody, calculate the inhibiting rate between each monoclonal antibody, and inhibiting rate is (1-measured value OD/ negative control OD) * 100; Inhibiting rate 〉=75% is for relevant, and 〉=50% is not exclusively relevant, and≤50% is uncorrelated, and≤25% for uncorrelated fully.Table 2 results suggest, three incomplete related antigen relevant ranges of 6 strain monoclonal antibodies identification.3, monoclonal antibody class and subclass are measured:
The 30ml cells and supernatant added 4 ℃ of solid ammonium sulfates 2 hours by 25.8%, and 3,000 left the heart 30 minutes, and solution is with the neutral PBS dissolving of 0.1ml.1% agarose, PBS does immune double diffusion with the anti-mouse blood grouping serum of rabbit (Sigma), observes precipitation line after 24 hours.Monoclonal antibody is IgG1 as a result.4, the analysis in monoclonal antibody identification native antigen site:
The same solid phase antigen, 50ulHCV core area antibody positive patients serum, the monoclonal antibody of 50ulHRP mark, 37 ℃ 2 hours.The same colour developing liquid that adds is surveyed the 492nm absorption value, and the positive serum inhibiting rate calculates the same site and analyzes; 〉=50% inhibition is positive.16 parts of HCV core area antibody positive serum have all shown positive inhibiting rate in various degree to three groups of antibody as a result, show that thus the antibody after the natural infection can suppress the reaction of monoclonal antibody and artificial antigen, therefore points out this site that three strains monoclonal antibody is discerned also to be present on the native antigen and (sees Table 3).(2) anti-HCV film district monoclonal antibody: 1, specific assay:
Measure with the same immunizing potency assay method of the antigen of different zones.The result shows outside antibody and the film district antigen-reactive not have specific reaction (seeing Table 1) with other regional antigen of HCV and contrast antigen.2, the monoclonal antibody recognition site is measured:
5 μ g/ml antigen 0.1ml/ holes add 96 orifice plates, 37 ℃ of solid phases are after 2 hours, the monoclonal antibody 50 μ l that add odd contradictive hydroperitoneum 50 μ l and HRP mark, 37 ℃ after 1 hour, add substrate solution, survey the 492nm absorption value, with odd contradictive hydroperitoneum the monoclonal antibody with-HRP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculate the inhibiting rate between each monoclonal antibody with known irrelevant odd contradictive hydroperitoneum, and inhibiting rate is (1-measured value OD/ negative control OD) * 100; Inhibiting rate 〉=75% is for relevant, and 〉=50% is not exclusively relevant, and≤50% is uncorrelated, and≤25% for uncorrelated fully, two incomplete related antigen relevant bits districts of 4 strain monoclonal antibodies identification as a result.3, monoclonal antibody class and subclass are measured:
The 30ml cells and supernatant added 4 ℃ of solid ammonium sulfates 2 hours by 25.8%, and 3,000 left the heart 30 minutes, and solution is with the neutral PBS dissolving of 0.1ml.1% agarose, PBS does immune double diffusion with the anti-mouse blood grouping serum of rabbit (Sigma), observes precipitation line after 24 hours.All antibody are mouse IgG1 as a result.(3) anti-HCVNS3 district monoclonal antibody: 1, specific assay:
Measure with the same immunizing potency assay method of the antigen of different zones.Outside result and the NS3 district antigen-reactive, there is not specific reaction (seeing Table 1) with other regional antigen of HCV and contrast antigen.2, the monoclonal antibody recognition site is measured:
5ug/ml antigen 0.1ml/ hole adds 96 orifice plates, 37 ℃ of solid phases are after 2 hours, the monoclonal antibody 50ul that adds odd contradictive hydroperitoneum 50ul and HRP mark, 37 ℃ after 1 hour, add substrate solution, survey the 492nm absorption value, with odd contradictive hydroperitoneum the monoclonal antibody with-HRP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculate the inhibiting rate between each monoclonal antibody with known irrelevant odd contradictive hydroperitoneum, promptly inhibiting rate is (1-measured value OD/ negative control OD) * 100; Inhibiting rate 〉=75% is for relevant; 〉=50% is not exclusively relevant, and≤50% is uncorrelated, and≤25% for uncorrelated fully.Two the incomplete related antigen positions district of 5 strain monoclonal antibodies identification as a result.3, monoclonal antibody class and subclass are measured:
The 30ml cells and supernatant added 4 ℃ of solid ammonium sulfates 2 hours by 25.8%, and 3,000 left the heart 30 minutes, and solution is with the neutral PBS dissolving of 0.1ml.1% agarose, PBS observes precipitation line with the ashamed immune double diffusion of the anti-mouse blood grouping serum of rabbit (Sigma) after 24 hours.All monoclonal antibodies are mouse IgG1 as a result.4, the analysis in monoclonal antibody identification native antigen site:
The same solid phase antigen, 50ulHCVNS3 domain antibodies positive patients serum, the monoclonal antibody of 50ulHRP mark, 37 ℃ 2 hours.The same colour developing liquid that adds is surveyed the 492nm absorption value, and the clear inhibiting rate of positive green blood calculates the same site and analyzes; 〉=50% inhibition is positive.19 parts of HCVNS3 domain antibodies positive serums have all shown positive inhibiting rate in various degree to two groups of antibody as a result, show that thus the antibody after the natural infection can suppress the reaction of monoclonal antibody and artificial antigen, so point out this site that two groups of monoclonal antibodies are discerned also to be present on the native antigen.(4) anti-HCVNS4 district monoclonal antibody: 1, specific assay:
Measure with the same immunizing potency assay method of the antigen of different zones.Outside result's demonstration and the NS4 district antigen-reactive, there is not specific reaction (seeing Table 1) with other regional antigen of HCV and contrast antigen.2, the monoclonal antibody recognition site is measured:
5ug/ml antigen 0.1ml/ hole adds 96 orifice plates, 37 ℃ of solid phases are after 2 hours, the monoclonal antibody 50ul that adds odd contradictive hydroperitoneum 50ul and HRP mark, 37 ℃ after 1 hour, add substrate solution, survey the 492nm absorption value, with odd contradictive hydroperitoneum the monoclonal antibody with-HRP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculate the inhibiting rate between each monoclonal antibody with known irrelevant odd contradictive hydroperitoneum, promptly inhibiting rate is (1-measured value OD/ negative control OD) * 100; Inhibiting rate 〉=75% is for relevant; 〉=50% is not exclusively relevant, and≤50% is uncorrelated, and≤25% for uncorrelated fully.Two the incomplete related antigen positions district of 4 strain monoclonal antibodies identification as a result.3, monoclonal antibody class and subclass are measured:
The 30ml cells and supernatant added 4 ℃ of solid ammonium sulfates 2 hours by 25.8%, and 3,000 left the heart 30 minutes, and solution is with the neutral PBS dissolving of 0.1ml.1% agarose, PBS does immune double diffusion with the anti-mouse blood grouping serum of rabbit (Sigma), observes precipitation line after 24 hours.All monoclonal antibodies are mouse IgG1 as a result.4, the analysis in monoclonal antibody identification native antigen site:
The same solid phase antigen, 50ulHCVNS4 domain antibodies positive patients serum, the monoclonal antibody of 50ulHRP mark, 37 ℃ 2 hours.The same adding, show bag liquid survey 492nm absorption value, and the positive serum inhibiting rate calculates the same site and analyzes; 〉=50% inhibition is positive.The result shows: a part HCVNS4 domain antibodies positive serum has all shown positive inhibiting rate in various degree to two groups of antibody, show that thus the antibody after the natural infection can suppress the reaction of monoclonal antibody and artificial antigen, so the upright point of pointing out these two groups of monoclonal antibodies to discern is present on the native antigen also.(5) anti-HCVNS5 district monoclonal antibody: 1, specific assay:
Measure with the same immunizing potency assay method of the antigen of different zones.Result: outside NS5 district antigen-reactive, do not have specific reaction (seeing Table 1) with other regional antigen of HCV and contrast antigen.2, the monoclonal antibody recognition site is measured:
5ug/ml antigen 0.1ml/ hole adds 96 orifice plates, 37 ℃ of solid phases are after 2 hours, the monoclonal antibody 50ul that adds odd contradictive hydroperitoneum 50ul and HRP mark, 37 ℃ after 1 hour, add substrate solution, survey the 492nm absorption value, with odd contradictive hydroperitoneum the monoclonal antibody with-HRP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculate the inhibiting rate between each monoclonal antibody with known irrelevant odd contradictive hydroperitoneum, promptly inhibiting rate is (1-measured value OD/ negative control OD) * 100; Inhibiting rate 〉=75% is for relevant; 〉=50% is not exclusively relevant, and≤50% is uncorrelated, and≤25% for uncorrelated fully.Two the incomplete related antigen positions district of 3 strain monoclonal antibodies identification as a result.3, monoclonal antibody class and subclass are measured:
The 30ml cells and supernatant added 4 ℃ of solid ammonium sulfates 2 hours by 25.8%, and 3,000 left the heart 30 minutes, and solution is with the neutral PBS dissolving of 0.1ml.1% agarose, PBS does immune double diffusion with the anti-mouse blood grouping serum of rabbit (Sigma), observes precipitation line after 24 hours.The result shows that 3 strain monoclonal antibodies are mouse IgG1.4, the analysis in monoclonal antibody identification native antigen site:
The same solid phase antigen, 50ulHCVNS5 domain antibodies positive patients serum, the monoclonal antibody of 50ulHRP mark, 37 ℃ 2 hours.The same colour developing liquid that adds is surveyed the 492nm absorption value, and the positive serum inhibiting rate calculates the same site and analyzes; 〉=50% inhibition is positive.The result shows: Fen HCVNS5 domain antibodies positive serum has all shown positive inhibiting rate in various degree to two groups of antibody, show that thus the antibody after the natural infection can suppress the reaction of monoclonal antibody and artificial antigen, so point out the anti-site of being discerned of these two groups of first also to be present on the native antigen.Four, the anti-HCV core area of the applied research of monoclonal antibody () monoclonal antibody: 1, antigen measuring: 40ug/ml monoclonal antibody 0.01M PH9.6 CB 0.1ml/ hole, add serum, 0.1ml/ hole, 37 ℃ of monoclonal antibodies that add the HRP mark after 2 hours again, add colour developing liquid and survey the 192nm absorption value, make negative control with the ashamed liquid of worker, measured value/control value 〉=2.1 are judged to the positive.The result shows: measure 96 parts of high-risk human serums with first group of monoclonal antibody, 5 parts have shown positive findings, point out thus and have the antigen that can record out among the patients serum, also point out this monoclonal antibody that very big using value (seeing Table 6) is arranged simultaneously.2, TPPA:
9/ml antigen 0.01M PH9.6 CB 0.1ml/ hole adds 50 μ l human serum to be checked, and the same survey of 50ulHRP mark monoclonal antibody 492nm absorption value is calculated inhibiting rate, and 〉=50% is positive.The result: the enzyme labelling monoclonal antibody shows that only 96 parts of HCV antibody positive serum have 50% serum inhibition monoclonal antibody and antigenic reaction 〉=50%, these serum confirm all to contain anti-HCV core area monoclonal antibody through indirect ELISA, point out monoclonal antibody specificity height in HCV antigen/antibody combination detects thus, easy and simple to handle, certain application value (seeing Table 3) is arranged.
The applied research results suggest:we have prepared the specific monoclonal antibody of identification HCV natural antigen with the artificial antigen of the different sections of HCV, the monoclonal antibody that we obtained is not only in the HCV fundamental research, and in HCV antigen-antibody detection reagent very high using value arranged all.1, the monoclonal antibody of using a certain section can be analyzed the growth and decline situation of this antibody-like among the patients serum exactly, heals afterwards and the investigation of result for the treatment of with the development of understanding the state of an illness.2, variant regional monoclonal antibody mixes, and makes up antigen-antibody screening detection kit, and it is easy and simple to handle, fast; And specificity also improves a lot.3; Directly or indirectly measure or the metainfective virus antigen of localization of HCV with monoclonal antibody.4; Affinitive layer purification separates the HCV antigen composition in body fluid or the tissue behind the crosslinked Sepharose-4B of monoclonal antibody; And the analysis and research natural antigen replaces artificial antigen; Promotes the fundamental research of HCV; Improves the specificity of existing detection kit.5; Analyze country variant; Different areas so that the metainfective antigen of different the infected HCV with monoclonal antibody; The type of determining HCV is to improve the effect of HCV treatment.6; Analyze the infected with the monoclonal antibody of the same area identification different loci; Discern the antibody growth and decline situation in a certain site; With the immunne response situation of relation and the infected between understanding HCV and the infected.7; Prepare antiidiotypic antibody with specificity HCV monoclonal antibody; Be used as the preparation of vaccine and replace existing artificial antigen.8; Detect the not variation of synantigen composition of HCV among the group infection person with monoclonal antibody; To understand HCV variation situation.9; Have monoclonal antibody useful as HCV the infected's of neutralizing effect treatment; And high risk population's prophylactic treatment.10; The monoclonal antibody solid phase is concentrating and separating HCV virion on suitable carriers; To improve the possibility of HCV virus separation and Culture.11, inquire into the function of corresponding HCV gene regions encoded protein matter with known monoclonal antibody. table 1: anti-HCV monoclonal antibody component specific assay monoclonal antibody antigen: core space film district NS3 NS4 NS5JDSHCC39+---JDSHCC58+---JDSHCC81+---JDSHCC105+---JDSHCC123+---JUSHCC221+---JDSHCE-44-+---JDSHCE-56+---JDSHCE-125+---JDSHCE-246-+---JDSHCNS3-1+---JDSHCNS3-3--|--JDSHCNS3-6--+--JDSHCNS3-8+----JUSHCNS3-9--+--JDSHCNS4-93---+-JDSHCCBS4-40---+-JDSHCNS4-13--+-JDSHCNS5-1----+JDSHCNS52----+.
Table 2: monoclonal antibody is measured monoclonal antibody antigen to the core space antigen reactivity of separate sources: N.J C11 J.Core CP10 CP9JDSHCC39+++--JDSHCC58+++--JDSHCC81+++--JDSHCC105+++--JDSHCC123+++++JDSHCC221+++++N.J, C11, J.Core: be gene engineering antigen CP10, CP9: Japanese synthetic polypeptide antigen table 3: unexpectedly strive the inhibition method and measure Antibody Results inhibiting rate (%) positive serum (part) negative serum (part) 0-9 4 3410-19 7 1320-29 9 2430-39 6 1540-49 20 1050-59 28 0 〉=60 22 0: 60,/96 0/96
Table 4: film district monoclonal antibody is to the antigen reactive mensuration monoclonal antibody of different peptide sections antigen: peptide A peptide BJDSHGE-44--JDSHCE-246+-JDSHCE-56-+JDSHCE-125-+ table 5: anti-NS3 district monoclonal antibody recognition site is measured enzyme mark monoclonal antibody (inhibiting rate %) odd contradictive hydroperitoneum NS3-1 NS3-3 NS3-6 NS3-8 NS3-9NS3-1 90.5 87.3 94.2 0 68.8NS3-3 85.0 96.5 92.7 0 59.6NS3-6 91.7 95.1 89.7 0 66.2NS3-3 86.0 95.8 83.6 95.6 91.9NS3-9 91.2 94.0 97.6 0 86.3
Table 6:HCV antigen measuring is P/N ratio sample (part)≤1.0 351.1-2.0 56 〉=2.1 5 as a result
Meter: 5/96
Claims (3)
1, a kind of hybridoma cell strain, the CCTCC preserving number is C94006, it is characterized in that secreting the monoclonal antibody in anti-HCV NS3 district.
2, the monoclonal antibody in a kind of anti-HCV NS3 district, it is by hybridoma cell strain secretion of claim 1.
3, the application of the monoclonal antibody of claim 2 in preparation serology antigen detecting agent box, wherein this test kit also comprises anti-HCV core area antibody.
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| CA2316130A1 (en) * | 1999-08-19 | 2001-02-19 | Masanori Fukui | Method for detection or determination of hcv core antigens and reagent for detection or determination for use therein |
| CN100391469C (en) * | 2000-02-14 | 2008-06-04 | 三菱制药株式会社 | Remedies for hepatitis C |
| CN100376671C (en) * | 2005-04-30 | 2008-03-26 | 华中科技大学同济医学院附属同济医院 | Hybridoma cell strain for resisting hepatitis B virus surface antigen and monoclonal antibody thereof |
| CN100387709C (en) * | 2005-10-28 | 2008-05-14 | 中国科学院上海生命科学研究院 | Anti HBeAg monoclone antibody cell strain, preparation method and anti HBeAg monoclone antibody |
| CA2738027C (en) * | 2008-09-25 | 2018-12-04 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Monoclonal anti-claudin 1 antibodies for the inhibition of hepatitis c virus infection |
| US8951521B2 (en) * | 2010-08-30 | 2015-02-10 | Riken | Compounds having activity of suppressing activation of TGF-β receptor, method for screening of the compounds, and composition for preventing or treating disease caused by hepatitis C virus |
| CN102329378B (en) * | 2011-09-15 | 2012-10-17 | 山东大学齐鲁医院 | HCV (hepatitis C virus) core antigen and antibody thereof as well as hybridoma cell lines secreting antibody |
| CN107286237B (en) * | 2017-08-07 | 2020-04-03 | 广州泰诺迪生物科技有限公司 | Acquisition and application of anti-hepatitis C virus antibody |
| CN109666649B (en) * | 2018-12-29 | 2022-03-11 | 山东莱博生物科技有限公司 | Engineering cell strain for stably and efficiently expressing HCV NS3 protein antibody and application thereof |
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| WO1993004084A1 (en) * | 1991-08-21 | 1993-03-04 | Abbott Laboratories | Monoclonal antibodies to putative hcv ns5 proteins and methos for using same |
| WO1993004205A1 (en) * | 1991-08-21 | 1993-03-04 | Abbott Laboratories | Monoclonal antibodies to putative hcv e2/ns1 proteins and methods for using same |
| EP0537856A1 (en) * | 1991-10-15 | 1993-04-21 | Akzo Nobel N.V. | Monoclonal antibodies to hepatitis C virus |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO1993004084A1 (en) * | 1991-08-21 | 1993-03-04 | Abbott Laboratories | Monoclonal antibodies to putative hcv ns5 proteins and methos for using same |
| WO1993004205A1 (en) * | 1991-08-21 | 1993-03-04 | Abbott Laboratories | Monoclonal antibodies to putative hcv e2/ns1 proteins and methods for using same |
| EP0537856A1 (en) * | 1991-10-15 | 1993-04-21 | Akzo Nobel N.V. | Monoclonal antibodies to hepatitis C virus |
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