CN102071169A - Hybridoma of DON (Deoxynivalenol) monoclonal antibody as well as preparation method and application thereof - Google Patents
Hybridoma of DON (Deoxynivalenol) monoclonal antibody as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the biological engineering field, in particular to hybridoma of a DON (Deoxynivalenol) monoclonal antibody as well as a preparation method and application thereof. Through cell fusion, an immunizing antigen and a detection antigen adopt DON-BSA (Bovine Serum Albumin) to obtain and store hybridoma secreting the DON monoclonal antibody with high efficiency, and the antibody has high sensitivity, strong specificity and appetency with DON and low cross reaction with other toxins. The invention lays the foundation for researching DON test reagents.
Description
Technical field:
The present invention relates to bioengineering field, relate to a kind of anti-deoxynivalenol (deoxynivalenol, DON) hybridoma of monoclonal antibody and its production and application especially.
Background technology:
In many pollution factors of agricultural products in China, mycotoxins is the important natural pollutant of a class, is the focus that the whole world is paid close attention to by its food origin disease that causes and food trade controversial issue always.Its toxic is big, the closest, the heaviest to crop pollution with the human health relation, the widest mycotoxins that distributes is deoxynivalenol (Deoxynivalenol, DON).Deoxynivalenol (DON) is not only the main toxin that the head blight wheat pollutes, but also can pollute cereal such as corn, barley, Chinese sorghum, people and animals' feed can be caused poisoning behind the grain of this endotoxin contamination and the feed, the sickle-like bacteria that produces DON still causes the The main pathogenic fungi of rice bakanae disease, vegetables farm crop such as (as joint melon joint cucurbit wilt), therefore DON pollutes and is not only an important food-safety problem, and is an important factor that causes crop production reduction, quality to descend on agricultural.So anti-DON monoclonal antibody to develop, create fast, sensitive, the detection method of DON accurately, not only to ensuring food safety, promote that human health is significant, and, improve agricultural output and quality assurance aspect and have vital role in the screening of high-quality, anti-bakanae disease paddy rice and vegetable variety.
The key that DON pollutes control is on-the-spot quick, dynamic monitoring toxin level, find out " resources " that toxin pollutes in each agricultural products, and at present China detects that above-mentioned toxin method exists that sensitivity is low, complex operation, contact severe toxicity standard substance, detection method are not suitable in batches sample preparation, be difficult to satisfy the on-the-spot shortcoming such as requirement that detects, and become " bottleneck " that restricts China's foodstuffs industry fast development and export of farm produce trade, press for quick, sensitive, as to be suitable for enterprise inspection by oneself and the basic unit's scene use DON immunoassay technology method of setting up.The immunization method that detects DON at present has enzyme-linked immunosorbent assay (ELISA), immune colloid gold method and chemical luminous immune detection method (CLIA).The key of this several method is the monoclonal antibody at DON of preparation high specificity, and utilize the antibody of high-affinity and the specific recognition between the antigen based on this, filter out best immunologic detection method and stdn, thus develop conveniently, economical, sensitive, fast, be suitable for basic unit and the on-the-spot immunity detection reagent that uses.
Because DON is haptens, therefore have only immunoreactivity (with the specific antibody reaction) and non-immunogenicity (can not stimulate body to produce antibody), must with the macromolecular carrier coupling after can stimulate body to produce antibody.The complete antigen that is used for immune animal when being reported at present preparation DON monoclonal antibody both at home and abroad is DON-ovalbumin (OVA) conjugate, and the envelope antigen that detects usefulness is DON-bovine serum albumin (BSA) conjugate.Do not appear in the newspapers both at home and abroad and this research immune animal and monoclonal antibody are all used conjugate (DON-BSA) with a kind of albumen and DON when detecting, adopt the carrier exclusive method to prepare anti-DON monoclonal antibody method.Owing to only need synthetic a kind of complete antigen, though so this method have economy, simple, timesaving characteristics, immunity and detect with method requirement height, the difficulty of different complete antigens big technically.
Cytogamy is passed through in this research, immunizing antigen and detection antigen all adopt DON-BSA, obtained the hybridoma cell strain of the anti-DON monoclonal antibody of efficient secretion, this antibody is highly sensitive, high specificity, with DON avidity height, low with other toxin cross reactions, for the development of DON detection reagent is laid a good foundation.
Summary of the invention:
(1) anti-DON MONOCLONAL ANTIBODIES SPECIFIC FOR
1. animal immune
With BSA, DON respectively with 1,3 two cyclic group diimine (DCC) and N-hydroxy-succinamide (NHS) coupling, by pentanedioic acid the two is connected to form the DON-BSA conjugate, adopt low dose of macrocyclic immunization protocol.
2. cytogamy
Sp2/0 cell and mouse boosting cell were mixed in the aseptic centrifuge tube of 50mL by 1: 10, the centrifugal 10min of 1000r/min, abandon supernatant, flicking pipe fully scatters cell in the end, centrifuge tube is put in 37 ℃ of water-baths, slowly added the 50%PEG0.7mL of 37 ℃ of pre-temperature, add in the 1min, leave standstill 90s in 37 ℃, slowly add the effect that the 20mL RPMI-1640 stops PEG.The centrifugal 10min of 800r/min abandons supernatant liquor, selects substratum (Gibco company product) the sedimentary cell that suspends with the HAT that contains 20% calf serum, and adjusting cell concn is 2 * 10
6/ mL is inoculated in respectively in 96 well culture plates that are added with feeder cell then, puts 37 ℃, cultivate in the 5%CO2 incubator, after merging 7～14d, use HT nutrient solution (Gibco company product) instead, day by day the observation of cell growing state, wait to grow to 1/3～1/2 visual field, the ELISA method detects antibody in the supernatant liquor.
3. hybridoma screening and antibody test
This research adopts the carrier exclusive method different with traditional method to carry out.Promptly be respectively envelope antigen, with anti-DON monoclonal antibody in the indirect elisa method screening fused cell supernatant, with the specificity of DON toxin indirect competition inhibition ELISA method conclusive evidence monoclonal antibody with 0.5 μ g/ml DON-BSA, BSA.With the positive contrast of the serum of immune mouse, be blank with the supernatant liquor of Sp2/0 cell cultures.
4. hybridoma cloning
ELISA and DON toxin indirect competition inhibition ELISA method detect supernatant male hole, and limiting dilution assay carries out cloning, to all mono-clonal hole supernatant antibody positive rate be 100%, with the cell enlarged culturing and build strain, that hybridoma is frozen in liquid nitrogen container.Send State Intellectual Property Office to specify patent microbial preservation center preservation simultaneously, preserving number is CGMCCNO.4107.
5. the production of monoclonal antibody: adopt the interior ascites of animal body to induce method.
6. Purification of Monoclonal Antibodies: adopt the saturated ammonium sulphate method that ascites is carried out purifying.
(2) evaluation of anti-DON monoclonal antibody
1. the immunoglobulin class of antibody and subgroup identification: adopt the antibody subclass to measure test kit and measure.
2. IgG antibody Determination on content: the ascites after adopting the BCA protein determination kit to sad-ammonium sulfate method purifying is carried out the IgG assay.
3. the mensuration of antibody molecule amount: adopt the SDS-polyacrylamide gel electrophoresis.
4. antibody titers is measured: with 0.5 μ g/ml DON-BSA is envelope antigen, and antibody is carried out doubling dilution, and with the negative contrast of Sp2/0 cell ascites refined solution, PBS is a blank, measures antibody titers in the purifying ascites with indirect ELISA method, with (A
Experiment-A
Blank)/(A
Negative-A
BlankThe maximum dilution multiple of) 〉=2.1 is the titration end point of antibody.The extent of dilution that the A value is about 1 o'clock antibody is the reference work extent of dilution.
5. the mensuration of affinity of antibody: adopt indirect elisa method, promptly measure the affinity costant of antibody.
6. the specific assay of antibody: measure with indirect competition inhibition ELISA method, the toxin of participating in the experiment is toxin such as NIV, DAS, T-2 toxin, F-X.
Adopt the present invention to obtain following technique effect:
Obtained immunne response preferably with DON-BSA immunity Balb/c mouse.After the cytogamy, through indirect non-competing ELISA method screening, and prove conclusively with the indirect competitive ELISA method, therefrom selecting antagonism DON toxin has the cell strain of inhibition, through 3～4 subclones, reach 3 times 100% positives, set up the hybridoma cell strain of 1 strain energy stably excreting DON monoclonal antibody, called after 3G5.
Suppress ELISA with indirect competition and record linearity range 9.8 μ g/L-10000 μ g/L, linear equation Y=-0.2726X+0.2259 (r=0.9309), minimum detectable concentration to DON is 9.2 μ g/L (seeing Figure of description 1), be higher than far away in the GB in the food≤750 μ g/L, in the feed≤requirement of the detection sensitivity of 1000 μ g/L.This shows that this is studied BSA, DON respectively with DCC and NHS coupling, by pentanedioic acid the two is connected to form the DON-BSA conjugate, this method coupling efficiency height, coupled antigen is stable, solvability is good, immunogenicity is strong.The BSA-DON conjugate not only is an immunizing antigen but also do detection antigen in this research simultaneously, prepared the hybridoma cell strain of the monoclonal antibody of secreting anti-DON with the method for cytogamy, compared with other haptens method for preparing monoclonal antibody, this method is simple, economically feasible, the antibody screening difficulty is big.The gained monoclonal antibody height of tiring, the anti-body contg height, the DON minimum detectable concentration is 9.2 μ g/L, highly sensitive, have high affinity with DON, low with other mycotoxins cross reaction, high specificity can be used for preparing the homemade DON-ELISA detection kit of high quality.
Description of drawings:
Figure of description 1 is a DON indirect competitive ELISA typical curve
Embodiment:
(1) anti-DON MONOCLONAL ANTIBODIES SPECIFIC FOR
1. animal immune
With BSA, DON respectively with 1,3 two cyclic group diimine (DCC) and N-hydroxy-succinamide (NHS) coupling, by pentanedioic acid the two is connected to form the DON-BSA conjugate, DON and BSA mol ratio are 50: 1.This method coupling efficiency height, coupled antigen is stable, and solvability is good, and immunogenicity is strong.
The DON-BSA conjugate is mixed with 1mg/ml solution, adopt low dose of macrocyclic immunization protocol, to the female Balb/c mouse in 6～8 ages in week (body weight 18～20g), first immunisation is with 100 μ g DON-BSA, add the complete freund adjuvant of equivalent (Sigma company product), abdominal injection.After 2 weeks,, add the incomplete freund adjuvant of equivalent (Sigma company product), abdominal injection with 100 μ g DON-BSA.After 2 weeks,, add the incomplete freund adjuvant of equivalent, abdominal injection again with 100 μ g DON-BSA.After 2 weeks, reinforced immunological, 50 μ g DON-BSA solution immunity in the spleen, extracting spleen cell merges behind the 4d.
2. cytogamy
Sp2/0 cell and mouse boosting cell were mixed in the aseptic centrifuge tube of 50mL by 1: 10, the centrifugal 10min of 1000r/min, abandon supernatant, flicking pipe fully scatters cell in the end, centrifuge tube is put in 37 ℃ of water-baths, slowly added the 50%PEG0.7mL of 37 ℃ of pre-temperature, add in the 1min, leave standstill 90s in 37 ℃, slowly add the effect that the 20mL RPMI-1640 stops PEG.The centrifugal 10min of 800r/min abandons supernatant liquor, selects substratum (Gibco company product) the sedimentary cell that suspends with the HAT that contains 20% calf serum, and adjusting cell concn is 2 * 10
6/ mL is inoculated in respectively in 96 well culture plates that are added with feeder cell then, puts 37 ℃, cultivate in the 5%CO2 incubator, after merging 7～14d, use HT nutrient solution (Gibco company product) instead, day by day the observation of cell growing state, wait to grow to 1/3～1/2 visual field, the ELISA method detects antibody in the supernatant liquor.
3. hybridoma screening and antibody test
This research adopts the carrier exclusive method different with traditional method to carry out.Promptly be respectively envelope antigen, with anti-DON monoclonal antibody in the indirect elisa method screening fused cell supernatant, with the specificity of DON toxin indirect competition inhibition ELISA method conclusive evidence monoclonal antibody with 0.5 μ g/ml DON-BSA, BSA.With the positive contrast of the serum of immune mouse, be blank with the supernatant liquor of Sp2/0 cell cultures, not grow clone's the negative contrast of cells and supernatant in the fused cell culture plate, the criterion of positive cell hole is (A
Test-A
Blank)/(A
Negative control-A
Blank) 〉=2.1.
The immunizing antigen that adopts in this research is identical carrier BSA with detecting antigen, makes that the haptens MONOCLONAL ANTIBODIES SPECIFIC FOR is simpler, and is more economical, more saves time.
4. hybridoma cloning
ELISA and DON toxin indirect competition inhibition ELISA method detect supernatant male hole, and limiting dilution assay carries out cloning, to all mono-clonal hole supernatant antibody positive rate be 100%, with the cell enlarged culturing and build strain, that hybridoma is frozen in liquid nitrogen container.Send State Intellectual Property Office to specify patent microbial preservation center to contain simultaneously.
5. the production of monoclonal antibody: adopt the interior ascites of animal body to induce method.
6. Purification of Monoclonal Antibodies: adopt the saturated ammonium sulphate method that ascites is carried out purifying.
(2) evaluation of anti-DON monoclonal antibody
1. the immunoglobulin class of antibody and subgroup identification: adopt the antibody subclass to measure test kit and measure.
2. IgG antibody Determination on content: the ascites after adopting the BCA protein determination kit to sad-ammonium sulfate method purifying is carried out the IgG assay.
3. the mensuration of antibody molecule amount: adopt the SDS-polyacrylamide gel electrophoresis.
4. antibody titers is measured: with 0.5 μ g/ml DON-BSA is envelope antigen, and antibody is carried out doubling dilution, and with the negative contrast of Sp2/0 cell ascites refined solution, PBS is a blank, measures antibody titers in the purifying ascites with indirect ELISA method, with (A
Experiment-A
Blank)/(A
Negative-A
BlankThe maximum dilution multiple of) 〉=2.1 is the titration end point of antibody.The extent of dilution that the A value is about 1 o'clock antibody is the reference work extent of dilution.
5. the mensuration of affinity of antibody: adopt indirect elisa method, promptly measure the affinity costant of antibody.The DON-BSA bag of getting three kinds of doubling dilution degree (0.25 μ g/mL, 0.50 μ g/mL, 1.00 μ g/mL) is added the purifying ascites of the concentration known of serial dilution by the polystyrene microplate, adopts indirect elisa method, measures the affinity costant of each antibody.With the A value purifying ascites concentration is drawn out 3 and measure curve, the A value that is tending towards flat sections with each curve top is 100%, protein concentration [Ab] t that to find its A value be 50% time point, can get [Ab] t, [Ab '] t and [Ab "] three values of t like this; be calculated as follows the K value then: when the envelope antigen concentration ratio is 2, K
1=1/2 (2[Ab '] t-[Ab] t), or K
2=1/2 (2[Ab "] t-[Ab '] t); When the envelope antigen concentration ratio is 4, K
3=3/2 (4[Ab "] t-[Ab] t).Get final product 3 K values, averaging is its affinity constant
6. the specific assay of antibody: measure with indirect competition inhibition ELISA method, the toxin of participating in the experiment is toxin such as NIV, DAS, T-2 toxin, F-X.
The subclass of antibody, relative molecular weight, antibody titers, affinity constant etc. see Table 1.Record the cross reacting rate of toxin such as anti-DON monoclonal antibody and NIV, DAS, T-2 toxin, F-X with indirect competition ELISA method, see Table 2.
The anti-DON monoclonal anti of table 1 bulk properties
The cross reacting rate of the anti-DON monoclonal antibody of table 2 and other analogue
(3). determining of indirect competitive ELISA testing condition
Through repeatedly experiment, conclusive evidence DON-BSA is best, and bag is 0.5 μ g/mL by concentration, package amount 100 μ l/ holes; The best effort concentration of anti-DON monoclonal antibody 1: 64000.Suppress ELISA with indirect competition and record linearity range 9.8 μ g/L-10000 μ g/L, linear equation Y=-0.2726X+0.2259 (r=0.9309), minimum detectable concentration to DON is 9.2 μ g/L (seeing Figure of description 1), be higher than far away in the GB in the food≤750 μ g/L, in the feed≤requirement of the detection sensitivity of 1000 μ g/L.
The biological material specimens explanation:
(1) depositary institution's title: China Committee for Culture Collection of Microorganisms common micro-organisms center
(2) depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
(3) preservation date: on August 30th, 2010
(4) deposit number: CGMCC NO.4107
(5) classification name: hybridoma cell strain
Claims (7)
1. monoclonal hybridoma of anti-deoxynivalenol, it is characterized in that: the preserving number of described cell is CGMCC NO.4107.
2. anti-deoxynivalenol monoclonal antibody is characterized in that: the cell of hybridoma that by preserving number is CGMCC NO.4107 is secreted, and the DON minimum detectable concentration is 9.2 μ g/L.
3. monoclonal preparation method of anti-deoxynivalenol is characterized in that comprising the next coming in order step:
1) conjugate preparation
BSA, DON respectively with 1,3 two cyclic group diimine and N-hydroxy-succinamide coupling, by pentanedioic acid the two is connected to form the DON-BSA conjugate, DON and BSA mol ratio are 50: 1; The DON-BSA conjugate is mixed with 1mg/ml solution, adopts low dose of macrocyclic immunization protocol, to the female mice in 6～8 ages in week; First immunisation adds the complete freund adjuvant of equivalent with 100 μ g DON-BSA; After 2 weeks,, add the incomplete freund adjuvant of equivalent with 100 μ g DON-BSA; After 2 weeks,, add the incomplete freund adjuvant of equivalent again with 100 μ g DON-BSA; After 2 weeks, reinforced immunological, 50 μ g DON-BSA solution immunity in the spleen, extracting spleen cell merges behind the 4d;
2) cytogamy
Sp2/0 cell and mouse boosting cell were mixed in the aseptic centrifuge tube of 50mL by 1: 10, and the centrifugal 10min of 1000r/min abandons supernatant, flicks pipe and the end cell is fully scattered; Centrifuge tube is put in 37 ℃ of water-baths, slowly added the 50%PEG 0.7mL of 37 ℃ of pre-temperature, add in the 1min; Leave standstill 90s in 37 ℃, slowly add the effect that the 20mL RPMI-1640 stops PEG; The centrifugal 10min of 800r/min abandons supernatant liquor, selects the substratum sedimentary cell that suspends with the HAT that contains 20% calf serum, and adjusting cell concn is 2 * 10
6/ mL is inoculated in respectively in 96 well culture plates that are added with feeder cell then, puts 37 ℃, cultivates in the 5%CO2 incubator, uses the HT nutrient solution instead after merging 7～14d; Day by day the observation of cell growing state waits to grow to 1/3～1/2 visual field, and the ELISA method detects antibody in the supernatant liquor;
3) hybridoma screening and antibody test
Be respectively envelope antigen with 0.5 μ g/ml DON-BSA, BSA, with anti-DON monoclonal antibody in the indirect elisa method screening fused cell supernatant, with the specificity of DON toxin indirect competition inhibition ELISA method conclusive evidence monoclonal antibody; With the positive contrast of the serum of immune mouse, be blank with the supernatant liquor of Sp2/0 cell cultures, not grow clone's the negative contrast of cells and supernatant in the fused cell culture plate;
4) hybridoma cloning
ELISA and DON toxin indirect competition inhibition ELISA method detect supernatant male hole, limiting dilution assay carries out cloning, through 3～4 subclones, reach 3 times 100% positives, set up the hybridoma cell strain of 1 strain energy stably excreting DON monoclonal antibody, preserving number is CGMCCNO.4107, and the secreted monoclonal antibody DON minimum detectable concentration of this hybridoma is 9.2 μ g/L.
4. monoclonal antibody as claimed in claim 1 is characterized in that: the application in detecting rice bakanae disease or joint cucurbit wilt.
5. hybridoma as claimed in claim 2 is characterized in that: the application in detecting rice bakanae disease or joint cucurbit wilt.
6. monoclonal antibody as claimed in claim 1 is characterized in that: detecting the farm crop Application in Food.
7. hybridoma as claimed in claim 2 is characterized in that: detecting the farm crop Application in Food.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792347A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Special test box for enzyme-linked immunosorbent assay of class-B trichothecenes |
| CN103808934A (en) * | 2012-11-07 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Preparation of ELISA kit for detecting deoxynivalenol |
| CN114051415A (en) * | 2019-07-03 | 2022-02-15 | 英特维特国际股份有限公司 | Conjugated deoxynivalenol for preventing mycotoxin poisoning |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6537762B1 (en) * | 1999-07-30 | 2003-03-25 | Board Of Trustees Of Michigan State University | Peptide mimotope to mycotoxin deoxynivalenol and uses thereof |
| CN101482566A (en) * | 2009-01-19 | 2009-07-15 | 南昌博恒生物制品有限公司 | Production and use of test paper for fast detecting deoxynivalenol in cereal |
-
2010
- 2010-09-09 CN CN2010102770205A patent/CN102071169B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6537762B1 (en) * | 1999-07-30 | 2003-03-25 | Board Of Trustees Of Michigan State University | Peptide mimotope to mycotoxin deoxynivalenol and uses thereof |
| CN101482566A (en) * | 2009-01-19 | 2009-07-15 | 南昌博恒生物制品有限公司 | Production and use of test paper for fast detecting deoxynivalenol in cereal |
Non-Patent Citations (3)
| Title |
|---|
| 《Journal of Agricultural Food Chemistry》 19881231 Casale等 Enzyme-Linked Immunosorbent assay employing monoclonal antibody specific for deoxynivalenol (vomitoxin) and severla analogues 663-668 1-7 第36卷, 第3期 2 * |
| 《中国农业科学》 20071231 李 华等 赤霉毒素脱氧雪腐镰刀菌烯醇(DON)酶联免疫检测方法研究 721-726 1-7 第40卷, 第4期 2 * |
| 《微生物学报》 20080604 祭芳等 抗脱氧雪腐镰刀菌烯醇单克隆抗体的制备 929-934 1-7 第48卷, 第7期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103792347A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Special test box for enzyme-linked immunosorbent assay of class-B trichothecenes |
| CN103808934A (en) * | 2012-11-07 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Preparation of ELISA kit for detecting deoxynivalenol |
| CN114051415A (en) * | 2019-07-03 | 2022-02-15 | 英特维特国际股份有限公司 | Conjugated deoxynivalenol for preventing mycotoxin poisoning |
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