CN101376677A - Monoclonal antibody IE5 for detecting new castle disease virus variation strain - Google Patents

Monoclonal antibody IE5 for detecting new castle disease virus variation strain Download PDF

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CN101376677A
CN101376677A CNA2008101402976A CN200810140297A CN101376677A CN 101376677 A CN101376677 A CN 101376677A CN A2008101402976 A CNA2008101402976 A CN A2008101402976A CN 200810140297 A CN200810140297 A CN 200810140297A CN 101376677 A CN101376677 A CN 101376677A
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monoclonal antibody
cell
ndv
strain
virus
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张秀美
胡北侠
黄艳艳
何叶峰
刘玉庆
张伟
颜世敢
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Institute Animal Science and Veterinary Medicine of Shandong AAS
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Abstract

The invention provides a monoclonal antibody for detecting new-castle disease virus variant. The preparation method comprises the following steps: immunizing a BALB/C mouse of eight ages using purified new-castle disease virus NDV LaSota strain as immunogen, boostering immnunization, standing for three days, and taking mouse splenic cells to fuse with myeloma cells SP2/0 in a logarithmic growth phase. The purified NDV LaSota strain is antigen coated enzyme marking board for screening positive Hybridoma cell, and the monoclone 1E5 is positive via ELISA detection, and is subcloned, and then injected into an abdominal cavity of a sensitized BALB/C mouse of 8 to 10 ages for preparing ascites. The mab bioactivity identification result indicates that mab 1E5 ascites has ELISA titer of 100 multiply 2<10>, immunoglobulin subclass of IgG2a, HI titer of 14log2, and cell neutralization titer for NDV standard virulent strain F48E8 of 320. The monoclonal antibody can be used for antigenic variation research of the new-castle disease virus to monitor and forecast epidemic situation of new castle disease, and provide scientific theoretical basis for the effective prevention and control of the new castle disease.

Description

A kind of monoclonal antibody 1E5 that detects the Avian pneumo-encephalitis virus variant
Technical field
Patent of the present invention relates to the diagnostic reagent in the Vet Biotechnology field, specifically is a kind of monoclonal antibody 1E5 that detects the Avian pneumo-encephalitis virus variant.
Background technology
Newcastle disease (ND) is a kind of height contact, the deadly infectious disease of bird, brings serious economy loss for the aviculture of many countries.The cause of disease of newcastle disease is Avian pneumo-encephalitis virus (NDV), belongs to the member of Paramyxoviridae Rubulavirus, is a kind of sub-thread non-segmented negative RNA viruses with cyst membrane.Hemagglutinin-neuraminidase on NDV surface (HN) and fusion rotein (F) are main immune protective antigens, and pathogenic closely related with virus.HN albumen mainly mediates viral recognizing cells surface receptor, and F albumen then promotes the fusion of virus envelope and cytolemma, and HN albumen also participates in cell fusion process.Discover that HN is the glycoprotein of a complex function, blood clotting and neuraminic acid enzymic activity are arranged.1989, Iorio etc. find in the research of monoclonal antibody and HN albumen test, the HN protein molecular has 7 successive eclipsed HN antigen sites (4 on three-dimensional structure, 14,1,12,2,23,3), carry out hemagglutination-inhibition test and neuraminidase inhibition test with the monoclonal antibody in these 7 sites, wherein the monoclonal antibody that combines with 5 sites can both by the blocking-up cell receptor combine with virus particle and in the infectivity of NDV, 12, the monoclonal anti physical efficiency in 2 and 23 sites obviously suppresses the NA activity, these three sites and 1, the monoclonal antibody in 14 sites is adsorbed on the cell by blocking virus, and energy obvious suppression HA activity, and remaining site (3,4) monoclonal antibody had not both had the HA activity and had not had the NA activity yet.
Since nineteen twenty-six is separated to NDV first from the Indonesia Java, 4 ND have taken place in the world be very popular, and each ND is popular all can have new genotype to occur, show that NDV new antigenic variation may occur under the selective pressure of vaccine.In recent years, the result of study of domestic NDV molecular epidemiology shows, China popular NDV promptly has old genotype (as gene II type), new genotype (as gene VII type) is also arranged, yet, does the antigenicity of new genotype NDV morph? the different experiments chamber is owing to experiment condition, experiment strain difference, and there is very big-difference in result of study.Because monoclonal antibody is the homogeneous antibody at single antigenic determinat, so it can be used for location, the quantity of epitope and the research of antigenic variation of virus antigen epitope as probe.Since the eighties, along with the continuous development and perfection of monoclonal antibody technique, monoclonal antibody has been widely used in diagnosis, virus variation analysis, antigenic characteristic, protein structure and the functional study of ND.
Summary of the invention
The objective of the invention is by monoclonal antibody 1E5 and NDV standard virulent strain F48E8, vaccine strain LaSota and C30 and the strong malicious strain isolated of NDV at hemagglutination-inhibition test (HI), the difference of reacting in the virus neutralization tests, the antigenic variability of HN is studied, find that first 9/17 strong malicious strain isolated of NDV has a neutrality epitope (also being the blood clotting site simultaneously) to lack, the variation antigenic for NDV HN provides ample evidence, and also epidemiological study and the epidemic monitoring for ND provides technique means.
The preparation process of the monoclonal antibody 1E5 of detection Avian pneumo-encephalitis virus variant of the present invention is as follows:
1) animal immune
NDV LaSota strain with purifying is an immunogen, immune 6-8 BALB/C mice in age in week.The first immunisation adequately emulsified virus antigen abdominal injection of the Freund's complete adjuvant 6-8 BALB/C mice in age in week, the antigen amount is 50ug/; With Freund's incomplete adjuvant emulsive same antigen amount second immunisation, two Zhou Houyong do not add the same antigen amount abdominal injection booster immunization of adjuvant, merge after 3 days after two weeks.
2) merge
Get the immune balb/c mice splenocyte and merge with the myeloma cell SP2/0 that is in logarithmic phase, fusogen is PEG4000, and the kunming mice abdominal cavity cell is as feeder cell, and selective medium is HAT, merges back packing 96 porocyte culture plates, places CO 2Incubator is cultivated, after 5-7 days with the HT 1/2HAT substratum that swaps out, after 7-10 days with the HT HAT that swaps out.Observe every day, and the sucking-off supernatant detects when hybridoma grows to hole floorage 1/10.
3) screening of positive hybridoma cell and build strain: indirect ELISA is adopted in the screening of positive colony, and positive colony press the limiting dilution assay subclone three times, selects the stronger mono-clonal enlarged culturing of reaction at last and the strain cell is built in conduct.
4) preparation of odd contradictive hydroperitoneum: select age in 8-10 week BALB/C mice abdominal injection sterile liquid paraffin oil 0.5ml/ only, pneumoretroperitoneum injection in 7 days is in the positive hybridoma cell 500,000 of logarithmic phase/only.Observe every day to mouse web portion and obviously expand, extract ascites and centrifugal after get supernatant ,-20 ℃ of preservations are standby.
5) the monoclonal antibody specificity is identified: by 96 hole enzyme plates, indirect ELISA is identified the monoclonal antibody atopic with bags such as SPF chick embryo allantoic liquid, infectious bronchitis virus and avian influenza virus.
6) the monoclonal antibody indirect ELISA titer is measured: with the NDV LaSota strain bag of purifying by 96 hole enzyme plates, the 1%BSA sealing.Carry out 2 times of doubling dilution to 100 * 2 again after the monoclonal antibody 1E5 ascites 1:100 dilution 12
7) the monoclonal antibody blood clotting suppresses (HI) characteristic: carry out according to a conventional method.Be HI antigen with NDV (LaSota) at first, the HI that measures monoclonal antibody 1E5 tires.Be HI antigen with different N DV strain respectively again, carry out the HI test.
8) in the monoclonal antibody and CHARACTERISTICS IDENTIFICATION: adopt the cell neutralization test to carry out.
Virocyte median infective dose (TCID 50) mensuration: get 9-11 age in days SPF chicken embryo, prepare chick embryo fibroblast (CEF) according to a conventional method, cell numeration back dilution is the cell suspension of 800,000/ml, and packing 96 porocyte culture plates are cultivated 24h for 37 ℃.Get each strain virus liquid of NDV with 10 times of doubling dilutions of serum-free DMEM basal liquid, get 10 respectively -3-10 -108 extent of dilution inoculation CEF, the 50ul/ hole, each extent of dilution is inoculated 5 holes, establishes blank simultaneously.Cultivate 72h for 37 ℃, get cell culture fluid mensuration HA and tire, be judged to infection with HA 〉=2log2.Calculate the TCID of each strain by Reed-Muench method 50
Cell neutralization test: dilute with 1:10 with PBS after the monoclonal antibody 1E5 aseptically process, respectively with isopyknic 200TCID 50Virus liquid mixes, 37 ℃ of effect 45min, and the CEF of inoculation culture 24h cultivates 72h for 37 ℃, gets cell culture fluid mensuration HA and tires, and is judged to infection with HA 〉=2log2.
Monoclonal antibody 1E5 detects the method for NDV variant, and it is as follows to detect step:
1) at first measures the blood clotting valency (HA tires) of NDV, according to the HA preparation 4 unit viruses of tiring;
2) monoclonal antibody 1E5 ascites is got 25ul doubling dilution to 2 after diluting with 1:10 with 0.01M PBS 11, add 25ul 4 unit viruses, room temperature effect 30min;
3) add 25ul 1% chicken erythrocyte suspension, room temperature effect 30min, result of determination is tired with HI〉4 log2 are judged to the positive, and HI tires≤and 4 log2 are judged to feminine gender.
Excellent effect of the present invention is: develop a kind of diagnostic reagent that can detect the NDV variant, finally provide a kind of effective technical means for large-scale clinical sample detects with the ND epidemic monitoring.
Embodiment:
1, the foundation of monoclonal antibody 1E5 hybridoma cell line:
NDV LaSota strain with purifying is an immunogen, and through cytogamy, the indirect ELISA screening obtains monoclonal antibody 1E5 positive colony, and it is carried out subclone three times, makes its ELISA positive rate reach 100%.Hybridoma is through frozen in liquid nitrogen container after the enlarged culturing.
2, age in the preparation of monoclonal antibody 1E5 ascites: 8-10 week BALB/C mice abdominal injection sterile liquid paraffin oil 0.5ml/ only, pneumoretroperitoneum injection in 7 days is in the positive hybridoma cell 500,000 of logarithmic phase/only.Observe every day to mouse web portion and obviously expand, extract ascites and centrifugal after get supernatant ,-20 ℃ of preservations are standby.Every mouse can be gathered in the crops 5ml left and right sides ascites.
3, the specific evaluation of monoclonal antibody 1E5 biology:
1) specificity is identified: with NDV, SPF chick embryo allantoic liquid, infectious bronchitis virus and avian influenza virus etc. is envelope antigen, and indirect ELISA is measured the specificity of monoclonal antibody 1E5.As a result monoclonal antibody 1E5 only with the NDV reacting positive, and all negative with reactions such as SPF chick embryo allantoic liquid, infectious bronchitis virus and avian influenza virus, show that monoclonal antibody 1E5 is the specific antibody at NDV.
2) blood clotting suppresses (HI) characteristic: with NDV LaSota is antigen, and it is 14 log2 that the HI of mensuration monoclonal antibody 1E5 tires.
3) in and characteristic: carry out with the cell neutralization test.
(1) get 9-11 age in days SPF chicken embryo, prepare chick embryo fibroblast (CEF) according to a conventional method, cell numeration back dilution is the cell suspension of 800,000/ml, and packing 96 porocyte culture plates are cultivated 24h for 37 ℃.Get NDV standard virulent strain F48E8 with 10 times of doubling dilutions of serum-free DMEM basal liquid, get 10 respectively -3-10 -108 extent of dilution inoculation CEF, the 50ul/ hole, each extent of dilution is inoculated 5 holes, establishes blank simultaneously.Cultivate 72h for 37 ℃, get cell culture fluid mensuration HA and tire, be judged to infection with HA 〉=2log2.Press the TCID that Reed-Muench method is calculated F48E8 50Be 10 -6.31/ 50ul.
(2) cell neutralization test: dilute with 1:10 with PBS after the monoclonal antibody 1E5 aseptically process, carry out 2 times of doubling dilution to 10 * 2 with PBS then 10, get each extent of dilution monoclonal antibody and isopyknic 200TCID respectively 50F48E8 virus liquid mixes, 37 ℃ of effect 45min, and the CEF of inoculation culture 24h cultivates 72h for 37 ℃, gets cell culture fluid mensuration HA and tires, and is judged to infection with HA 〉=2log2.With can in and 200TCID 50The highly diluted multiple of monoclonal antibody of virus liquid is that the neutralization of monoclonal antibody is tired.After testing, calculating that monoclonal antibody 1E5 tires to the neutralization of F48E8 is 320.
4. monoclonal antibody 1E5 is used for the analysis of NDV epidemic strain antigenic variability:
1) HI test: the HA that measures each NDV separation poison respectively tires, and prepares 4 unit viruses.Get the monoclonal antibody 1E5 ascites that 25ul10 doubly dilutes and carry out 2 times of doubling dilutions to 2 with PBS 11, each separates the 4 units virus liquid of poison, mixing, room temperature effect 30min to add 25ul respectively.Add 25ul 1% chicken erythrocyte suspension, mixing, room temperature effect 30min, result of determination.Monoclonal antibody 1E5 separates the reactivity of poison in HI and sees Table 1 with NDV.
2) neutralization test: adopt the cell neutralization test to carry out.
(1) get 9-11 age in days SPF chicken embryo, prepare chick embryo fibroblast (CEF) according to a conventional method, cell numeration back dilution is the cell suspension of 800,000/ml, and packing 96 porocyte culture plates are cultivated 24h for 37 ℃.Get NDV standard virulent strain F48E8 and separate poison with 17 NDV, get 10 respectively with 10 times of doubling dilutions of serum-free DMEM basal liquid -3-10 -108 extent of dilution inoculation CEF, the 50ul/ hole, each extent of dilution is inoculated 5 holes, establishes blank simultaneously.Cultivate 72h for 37 ℃, get cell culture fluid mensuration HA and tire, be judged to infection with HA 〉=2log2.Press the TCID that Reed-Muench method is calculated virus 50, the results are shown in Table 1.
(2) cell neutralization test: dilute with 1:10 with PBS after the monoclonal antibody 1E5 aseptically process, get monoclonal antibody respectively with isopyknic 200TCID 50Virus liquid mixes, 37 ℃ of effect 45min, and the CEF of inoculation culture 24h cultivates 72h for 37 ℃, gets cell culture fluid mensuration HA and tires, and is judged to infection with HA 〉=2log2.Test-results sees Table 1.
The detection step that monoclonal antibody 1E5 detects the NDV variant is as follows:
1) at first measures the blood clotting valency (HA tires) of NDV, according to the HA preparation 4 unit viruses of tiring;
2) monoclonal antibody 1E5 ascites is got 25ul doubling dilution to 2 after diluting with 1:10 with 0.01M PBS 11, add 25ul 4 unit viruses, room temperature effect 30min;
3) add 25ul 1% chicken erythrocyte suspension, room temperature effect 30min, result of determination is tired with HI〉4 log2 are judged to the positive, and HI tires≤and 4 log2 are judged to feminine gender.
Result of study of the present invention shows, monoclonal antibody 1E5 institute identified epitope is on the HN albumen on NDV surface, this site is during the blood clotting site also is simultaneously and site, and part NDV strain isolated loses the reactivity with monoclonal antibody 1E5 in HI and neutralization test, show that 1E5 identification antigen site lacks, and the variant of vaccine strain and standard virulent strain promptly occurred being different from the NDV epidemic strain on these strain isolateds.
The reactivity of the different strains of table 1 monoclonal antibody 1E5 in HI and neutralization test with NDV
Figure A200810140297D00081
Annotate: 1. "-" represents reaction negative, "+" expression reacting positive.2. this test is not carried out in " # " expression.

Claims (2)

1. monoclonal antibody 1E5 who detects the Avian pneumo-encephalitis virus variant, it is characterized in that with purified new-castle disease virus (NDV) LaSota strain be immunogen, immunity BALB/C mice in 8 age in week, got mouse boosting cell and the SP2/0 cytogamy that is in logarithmic phase behind the last booster immunization in 3 days, NDV LaSota strain with purifying is the screening that antigen coated enzyme plate is used for positive hybridoma cell, after ELISA detects male mono-clonal 1E5 and carries out 3 subclones, the 8-10 BALB/C mice abdominal cavity in age in week of injection sensitization prepares ascites, by monoclonal antibody biologic activity qualification result is shown, it is 100 * 2 that monoclonal antibody 1E5 ascites ELISA tires 10, immunoglobulin subclass is IgG2a, and it is 14 log2 that HI tires, and it is 320 that the cell neutralization of NDV standard virulent strain F48E8 is tired;
Concrete preparation process is as follows:
1) animal immune
NDV LaSota strain with purifying is an immunogen, immune 6-8 BALB/C mice in age in week, and the first immunisation adequately emulsified virus antigen abdominal injection of the Freund's complete adjuvant 6-8 BALB/C mice in age in week, the antigen amount is 50ug/; With Freund's incomplete adjuvant emulsive same antigen amount second immunisation, two Zhou Houyong do not add the same antigen amount abdominal injection booster immunization of adjuvant, merge after 3 days after two weeks;
2) merge
Get the immune balb/c mice splenocyte and merge with the SP2/0 cell that is in logarithmic phase, fusogen is PEG4000, and the kunming mice abdominal cavity cell is as feeder cell, and selective medium is HAT, merges back packing 96 porocyte culture plates, places CO 2Incubator is cultivated, and with the HT HAT that swaps out, observe every day after 7-10 days, and the sucking-off supernatant detects when hybridoma grows to hole floorage 1/10;
3) screening of positive hybridoma cell and build strain
Indirect ELISA is adopted in the screening of positive colony, and positive colony is pressed the limiting dilution assay subclone three times, selects the stronger mono-clonal enlarged culturing of reaction at last also as building the strain cell;
4) preparation of monoclonal antibody ascites
8-10 BALB/C mice abdominal injection sterile liquid paraffin 0.5ml/ in age in week only, the injection of 7 days pneumoretroperitoneums is in the positive hybridoma cell 500,000 of logarithmic phase/only, observe every day to mouse web portion and obviously expand, extract ascites and centrifugal after get supernatant ,-20 ℃ of preservations are standby;
5) monoclonal antibody 1E5 specificity is identified
Select for use SPF chick embryo allantoic liquid, infectious bronchitis virus and avian influenza virus bag by 96 hole enzyme plates, indirect ELISA is identified the monoclonal antibody specificity;
6) the monoclonal antibody indirect ELISA titer is measured
By 96 hole enzyme plates, the 1%BSA sealing is carried out 2 times of doubling dilution to 100 * 2 after the monoclonal antibody 1E5 ascites 1:100 dilution again with the NDV LaSota strain bag of purifying 12
7) the monoclonal antibody blood clotting suppresses (HI) CHARACTERISTICS IDENTIFICATION: carry out according to a conventional method; Be HI antigen with NDV LaSota at first, the HI that measures monoclonal antibody 1E5 tires, and is HI antigen with different N DV strain respectively again, carries out the HI test;
8) in the monoclonal antibody and CHARACTERISTICS IDENTIFICATION: adopt the cell neutralization test to carry out, concrete grammar is as follows:
Virocyte median infective dose (TCID 50) mensuration: get 9-11 age in days SPF chicken embryo, prepare chick embryo fibroblast CEF according to a conventional method, cell numeration back dilution is the cell suspension of 800,000/ml, packing 96 porocyte culture plates, cultivate 24h for 37 ℃, get each strain virus liquid of NDV with 10 times of doubling dilutions of serum-free DMEM basal liquid, get 10 respectively -3-10 -108 extent of dilution inoculation CEF, the 50ul/ hole, each extent of dilution is inoculated 5 holes, establishes blank simultaneously, cultivates 72h for 37 ℃, gets cell culture fluid mensuration HA and tires, and is judged to infection with HA 〉=2log2, calculates the TCID of each strain by Reed-Muench method 50
Cell neutralization test: dilute with 1:10 with PBS after the monoclonal antibody 1E5 aseptically process, respectively with isopyknic 200TCID 50Virus liquid mixes, 37 ℃ of effect 45min, and the CEF of inoculation culture 24h cultivates 72h for 37 ℃, gets cell culture fluid mensuration HA and tires, and is judged to infection with HA 〉=2log2;
2. monoclonal antibody 1E5 detects the method for NDV variant: it is as follows to it is characterized in that detecting step:
1) at first measures the blood clotting valency (HA tires) of NDV, according to the HA preparation 4 unit viruses of tiring;
2) monoclonal antibody 1E5 ascites is got 25ul doubling dilution to 2 after diluting with 1:10 with 0.01M PBS 11, add 25ul 4 unit viruses, room temperature effect 30min;
3) add 25ul 1% chicken erythrocyte suspension, room temperature effect 30min, result of determination is tired with HI〉4 log2 are judged to the positive, and HI tires≤and 4 log2 are judged to feminine gender.
CNA2008101402976A 2008-09-25 2008-09-25 Monoclonal antibody IE5 for detecting new castle disease virus variation strain Pending CN101376677A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102426258A (en) * 2011-11-11 2012-04-25 中国兽医药品监察所 Positive serum national standard substance used for newcastle disease hemagglutination inhibition test and preparation method thereof
CN105567644A (en) * 2014-03-13 2016-05-11 华中农业大学 Monoclonal antibodies capable of resisting Newcastle disease virus
CN110018304A (en) * 2019-05-15 2019-07-16 河南省农业科学院 A kind of newcastle epidemic disease antibody blocking Test paper
CN110669129A (en) * 2019-10-31 2020-01-10 江苏省农业科学院 Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102426258A (en) * 2011-11-11 2012-04-25 中国兽医药品监察所 Positive serum national standard substance used for newcastle disease hemagglutination inhibition test and preparation method thereof
CN102426258B (en) * 2011-11-11 2014-03-05 中国兽医药品监察所 Positive serum national standard substance used for newcastle disease hemagglutination inhibition test and preparation method thereof
CN105567644A (en) * 2014-03-13 2016-05-11 华中农业大学 Monoclonal antibodies capable of resisting Newcastle disease virus
CN110018304A (en) * 2019-05-15 2019-07-16 河南省农业科学院 A kind of newcastle epidemic disease antibody blocking Test paper
CN110669129A (en) * 2019-10-31 2020-01-10 江苏省农业科学院 Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof
CN110669129B (en) * 2019-10-31 2022-09-02 江苏省农业科学院 Newcastle disease virus HN protein monoclonal antibody 1G4 and application thereof

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