CN102994454B - Monoclonal antibody hybridoma cell strain of soybean lipoxygenase Lox3 as well as antibody and application of monoclonal antibody - Google Patents
Monoclonal antibody hybridoma cell strain of soybean lipoxygenase Lox3 as well as antibody and application of monoclonal antibody Download PDFInfo
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- CN102994454B CN102994454B CN201210554907.3A CN201210554907A CN102994454B CN 102994454 B CN102994454 B CN 102994454B CN 201210554907 A CN201210554907 A CN 201210554907A CN 102994454 B CN102994454 B CN 102994454B
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Abstract
The invention discloses a monoclonal antibody hybridoma cell strain of soybean lipoxygenase Lox3, wherein a collection number of the monoclonal antibody hybridoma cell strain is CGMCC5123. The invention further discloses a monoclonal antibody secreted by the hybridoma cell strain and application of the hybridoma cell strain to detection of the soybean lipoxygenase Lox3.
Description
Division explanation
The application's case is to be on August 2nd, 2011 applying date, and application number is 201110219334.4, the dividing an application of the monoclonal antibody hybridoma cell strain that denomination of invention is soybean fat oxidase isoenzyme and antibody thereof and application patent application.
Technical field
The present invention relates to a kind of monoclonal antibody hybridoma cell strain and monoclonal antibody thereof, be particularly related to monoclonal antibody hybridoma cell strain and the monoclonal antibody thereof of secreting respectively anti-soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3, cell strain deposit number is respectively: CGMCC5121, CGMCC5122 and CGMCC5123, also relate to the monoclonal antibody of each autocrine utilizing DASELISA immunoabsorption (DAS-ELISA) to detect the central application of soybean fat oxidase isoenzyme, belong to technical field of biological.
Background technology
In soybean, contain three kinds of lipoxidase (Lipoxygenase, abbreviation Lox) isozyme is Lox1, Lox2 and Lox3, they can single-minded catalysis have cis, cis-1, the polybasic unsaturated fatty acid of 4-pentadiene structure, by molecule oxygenation, forms the hydroperoxidation derivative with conjugated double bond, and aldehyde, ketone etc. have volatile smell substance, make soybean there is beany flavor, thereby reduced keeping quality, consumption and the nutritive value of bean product.Conventionally in soyabean processing process, people eliminate beany flavor by the methods such as pH value, organic solvent extraction and aldehyde lytic enzyme of heating, microwave irradiation, change medium, but these methods all can reduce the nutritive value of bean product to some extent, and make cost up.Cultivating lacking lipoxygenase kind can fundamentally address the above problem.But in the Breeding Process of lacking lipoxygenase material, in field, there is not indication trait in Lox, from seed outward appearance and economical character, cannot differentiate, and field lacking lipoxygenase material selects workload large, be therefore badly in need of a kind of quick, easy, cost is low, once can detect the detection method of three kinds of lipoxidases simultaneously.
The conventional detection method of lipoxidase is thin layer isoelectric focusing electrophoresis (IEF) detection method at present, this method is mainly to dye method and kinds of staining methods for protein in conjunction with Lox isozyme band is developed the color by enzyme, although can detect three kinds of lipoxidases, the anodal drift phenomenon of other Lox iso-electric point usually can occur because of the disappearance of Lox in detecting simultaneously; The method required equipment, reagent are expensive in addition, and operational requirement is strict, and general breeding, storage unit and processing quality testing department are awkward.
In addition according to Japanese Suzuki, report (the Prodution and use of monolional antibodies against rice embryo lipoxygenase-3 in 1992 such as Y, Biosci Biotech.Biochem.56:678-679): by monoclonal antibody, can detect the Lox3 in paddy rice, although this method result accurately and reliably, but in testing process except needs seal, also need the coated elisa plate that spends the night, therefore consuming time, inconvenience operates.
The patent of application number 00112539.7 discloses a kind of detection method of crop fat oxidase isoenzyme, the feature such as that the method has is easy and simple to handle, visual result.But because it is to utilize the principle of the coupled oxidation reduction indicator color reaction of lipoxidase to measure, so its sensitivity and specificity are subject to certain restrictions.
Summary of the invention
The object of the invention is to avoid the defect of aforesaid method, foundation can be secreted respectively 3 hybridoma cell strains of the monoclonal antibody specific of anti-soybean fat oxidase isoenzyme Lox1, Lox2, Lox3, and the monoclonal antibody specific that utilizes these cell strains to secrete is assembled can be for detect DASELISA immunosorption (DAS-ELISA) detection kit of three kinds of fat oxidase isoenzymes in soybean simultaneously.This test kit is applicable to the quantitative and qualitative analysis of three kinds of lipoxidases in soybean to measure.
First technical problem solved by the invention has been to provide the monoclonal antibody hybridoma cell strain of one group of anti-soybean fat oxidase isoenzyme, it is characterized in that described monoclonal antibody hybridoma cell strain comprises the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox1, splenocyte and the generation of SP2/0 cell process cytogamy for mouse, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC5121; And
The monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox2, splenocyte and the generation of SP2/0 cell process cytogamy for mouse, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC5122; And
The monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox3, splenocyte and the generation of SP2/0 cell process cytogamy for mouse, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC5123.
The establishment method of described anti-soybean fat oxidase Lox1, the monoclonal antibody hybridoma cell strain of Lox2, Lox3 is as follows:
1. immunizing antigen preparation: get respectively the soybean seeds (being designated as Lox-1) of single Lox1 of lacking, the soybean seeds (being designated as Lox-3) of the soybean seeds five-pointed star of single Lox2 of lacking No. 2 (being designated as Lox-2), single Lox3 of lacking (Lox-1, Lox-3 can obtain by national genebank, five-pointed star No. 2 (Lox-2) can have been bought from the market), with file, wear into bean powder, and the product Soybean Lox type1-B(of Sigma company contains 3 kinds of soybean fat oxidases simultaneously, hardly containing other soybean protein), respectively with 8～15 times of volumes the pH Tris-CaCl that is 8.0
2damping fluid mixes, lixiviate 0.5-1 hour, and 4 ℃ of centrifugal 10min of 12000rpm,, get supernatant standby, be respectively Lox-1 supernatant liquor, Lox-2 supernatant liquor, Lox-3 supernatant liquor, Soybean Lox type1-B supernatant liquor;
2. mouse immune: select the female mouse of pure lines BALB/C of the Quick off the mark in 4～6 week age, weigh respectively each Mouse Weight mark; Get step 1. Lox-1, Lox-2, the Lox-3 supernatant liquor of middle preparation, with physiological saline, be diluted to protein content 4mg/ml, after adding equal-volume Freund's complete adjuvant to mix, take subcutaneous multi-point injection first immunisation mouse, each supernatant liquor is immunity 5 mouse making marks respectively, and the immunizing dose of every mouse is 0.4～0.6mg;
3. inject endoxan solution (CY): by step 2. in the mouse of first immunisation respectively at immune 10min, 24h, 48h after, the endoxan solution (CY) that respectively mouse is carried out to the multi-point injection concentration 20mg/ml of nape portion according to the amount of 90mg/kg is subdued;
4. titration and again immunity: first immunisation after 1 week with step 1. in the Soybean Lox type1-B supernatant liquor of preparation do antigen to step the mouse in 3. according to step 2. method again carry out immunity, after immune one week for the second time, carry out immunity for the third time, after immunity, within the 4th day, from mouse tail, getting respectively blood 2-3 for the third time drips, centrifuging and taking supernatant, with indirect enzyme-linked immunosorbent assay, measure its tire (titration utilizes Soybean Lox type1-B to be coated with), if tired, be less than 1:1600, after 1 week to each mouse with Soybean Lox type1-B according to step 2. method again carry out immunity, and carry out titration, until serum titer reaches or higher than 1:1600,
5. booster immunization: in 3 kinds of immune mouses, every kind all select mouse that serum titer is the highest first 3 days of cytogamy by 4. booster immunization 1 time of step;
6. cytogamy: get respectively the splenocyte of different mouse and the SP2/0 cell of recovery of step in 5. and mix, screen respectively positive hybridoma cell on Tissue Culture Plate HAT substratum;
7. specificity positive cell strain screening: cytogamy is after 7～15 days, adopt indirect enzyme-linked immunosorbent assay, utilize Soybean Lox type1-B to carry out positive-selecting as detectable antigens, utilize the bean powder supernatant liquor of the soybean seeds (Lox-1) of single Lox1 of lacking to carry out cross detection as detectable antigens simultaneously, the ratio of the OD450 value of supernatant liquor and the OD450 value in blank hole be greater than 2.1 be judged as positive hole, and react the negative hole of performance with Lox-1, hybridoma is wherein called positive cell strain, the monoclonal antibody of this cell strain secretion is the anti-Lox1's of specificity.Use the same method, all utilize Soybean Lox type1-B to carry out positive-selecting as detectable antigens, utilize No. 2 bean powder supernatant liquors of five-pointed star to carry out cross detection to secreting the monoclonal antibody of anti-Lox2, utilize Lox-3 bean powder supernatant liquor to carry out cross detection to secreting the monoclonal antibody of anti-Lox3;
8. subclone: subclone is carried out respectively in 3 kinds of positive cell strains that adopt limiting dilution assay to screen in 7. step, after double subclone, weed out false positive cell strain, in testing process, all according to the method in 7., carry out the positive and cross detection simultaneously, finally can obtain the hybridoma cell strain of the monoclonal antibody specific of secreting respectively anti-Lox1, Lox2, Lox3.
Second technical problem solved by the invention is to provide the monoclonal antibody by monoclonal antibody hybridoma cell strain secretion claimed in claim 1, it is characterized in that described monoclonal antibody comprises the monoclonal antibody of the anti-Lox1 of specificity being secreted by the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox1; And
The monoclonal antibody of the anti-Lox2 of specificity being secreted by the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox2; And
The monoclonal antibody of the anti-Lox3 of specificity being secreted by the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox3.
Above-mentioned monoclonal antibody obtains by the following method: secretion Lox1, the Lox2 that above-mentioned steps is obtained in 8., Lox3 cell strain of monoclonal antibody are respectively with (1～2) * 10
6individual cell/amount is only inoculated into through the pretreated different B ALB/C mouse peritoneal of paraffin oil, and induction ascites produces antibody, after 6～8 days, can gather ascites or serum, obtains respectively Lox1, Lox2, Lox3 monoclonal antibody.
The 3rd technical problem solved by the invention is to provide the application of described monoclonal antibody in detecting soybean fat oxidase isoenzyme.
The 4th technical problem solved by the invention is to provide described monoclonal antibody and detects the application in soybean fat oxidase isoenzyme test kit in preparation.
The 5th technical problem solved by the invention is to provide a kind of method that detects soybean fat oxidase isoenzyme, it is characterized in that comprising following steps:
A, by each secreted monoclonal antibody separation and purification of the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox1, Lox2, Lox3 respectively of the present invention;
Monoclonal antibody after B, employing horseradish enzyme labelling steps A purifying, obtains respectively the beans Lox1 of the Chinese People's Anti-Japanese Military and Political College, the Lox2 of horseradish enzyme labelling and the monoclonal antibody of Lox3, as detecting antibody, and difference called after L-1 after enzyme mark, L-2, L-3;
C, utilize the polyclonal antibody of soybean fat oxidase isoenzyme Lox1, Lox2, Lox3 as insolubilized antibody coated elisa plate, the L-1 obtaining in B, L-2, L-3 is as detecting antibody, utilize the association reaction of antigen-antibody, by whether developing the color and the depth detects disappearance type and the content of Lox1, Lox2 in the middle of soybean seeds, these 3 kinds of lipoxidases of Lox3.
In described steps A, resulting each monoclonal antibody adopts caprylic acid-ammonium to carry out separation and purification, and step is as follows:
1. with 0.06M acetate buffer solution, gathered ascites or serum are diluted to 3-4 doubly, with NaOH, adjusting pH is 4.5, and writes down liquid volume;
2. under vibration, drip sad (in the ascites of every milliliter of dilution or serum, adding 25ul sad), under room temperature, stir 30min, 4 ℃, the centrifugal 30min of 10000rpm, abandons precipitation, stays supernatant; (adding sad must drop by drop adding)
3. filtering supernatant, adds the every 10 portions of supernatant liquors of PBS(of 0.1M pH7.4 to add 1 part of PBS), with NaOH, adjust pH to be 7.4,4 ℃ and place 30min;
4. grind ammonium sulfate, every milliliter of mixed solution adds 0.28g ammonium sulfate, stirs 30min, the centrifugal 20min of 10000rpm;
5. get precipitation and be dissolved in the 0.01M PBS of former ascites or serum 1/10 volume, by the 0.01MPBS damping fluid dialysed overnight of 50 times of volumes, change liquid 2-3 time;
6. liquid is taken out from dialysis tubing, 4 ℃, the centrifugal 20min of 10000rpm, goes precipitation, is the monoclonal antibody of purifying, and concentrated latter-20 ℃ save backup.
The step that the described beans Lox1 of the step B Zhong Jiang Chinese People's Anti-Japanese Military and Political College, Lox2 and the monoclonal antibody of Lox3 are carried out enzyme mark preparation detection antibody is as follows:
1. the HRP enzyme that takes 2mg, is dissolved in tri-distilled water, adds recently prepared sodium periodate solution, mixes rearmounted 4 ℃, and 30min, adds ethanolic soln, room temperature, and 30min, adds 1mg antibody purification, mixes, adjust pH to 9.0,4 ℃ are spent the night;
2. add sodium borohydride, mix, put 4 ℃ of 2h;
3. enzyme labelled antibody mixed solution is added to equal-volume saturated ammonium sulphate solution, put after 4 ℃ of 30min centrifugal, by pH7.4 phosphate buffered saline buffer dialysed overnight.
The step that in described step C prepared by soybean fat oxidase polyclonal antibody is as follows:
1. immune animal is selected and initial immunity: select the purebred new zealand white rabbit of weight between 2～3kg, Soybean Lox type1-B albumen is diluted to 8mg/ml as antigen, after isopyknic Freund's complete adjuvant mixing and emulsifying, take the subcutaneous multi-point injection immune rabbit in back, every rabbit immunizing dose is 1.6mg;
2. immunity again: immunity is again carried out according to step after utilizing afterwards the Soybean Lox type1-B albumen equal-volume mixing that incomplete Fu Shi adjuvant and concentration are 8mg/ml for 10～14 days in interval 1., so carries out immunity for the third time;
3. titration: for the third time after immunity the 11st day from above-mentioned steps 2. immune White Rabbit auricular vein get blood examination and survey it and tire, if tired while being less than 1:10000, to step 2. in White Rabbit again according to 2. immunity of step, so repeatedly immunity until survey and tire higher than 1:10000;
4. the preparation of polyclonal antibody: the White Rabbit of 3. tiring the highest to step after 3 days adopts the blood sampling of carotid artery depletion method, separation of serum, adopt indirect enzyme-linked immunosorbent assay to measure intersecting of other albumen in separation of serum and Lox1, Lox2, Lox3 and soybean, prove this serum and Lox1, Lox2, Lox3 all have intersect and with soybean in other albumen without obtaining polyclonal antibody after intersecting.
The disappearance type and the content that in described step C, detect soybean seeds central Lox-1, Lox-2, these 3 kinds of lipoxidases of Lox-3 comprise following operation steps:
1. the preparation of Lox extracting solution in soybean to be measured: the seed of soybean to be measured is worn into bean powder, the Tris-CaCl that the pH that adds 8～15 times of volumes is 8.0
2damping fluid, mixes rear centrifuging and taking supernatant standby;
2. wash plate: with Tween20-NaCl damping fluid, the coated enzyme plate of polyclonal antibody is washed 3～4 times repeatedly, each 2～3min, pats dry, and object washes away the polyclonal antibody not being attached on plank;
3.-1 application of sample: by step 1. in the Lox extracting solution of preparation be added in the enzyme plate that 2. step get ready, do the positive and negative control simultaneously, according to step, 2. wash plate after placing 30～90min for 25 ℃～37 ℃;
3.-2 application of samples: the reference liquid that sigma company Soybean Lox type1-B product is diluted to 800,000 unit of activity/ml, with Tween20-NaCl damping fluid, press 10, 30, 90, 270, 810, 2430, 7290, 21870, 65610, 196830, 590490, 0 totally 12 weaker concns carry out doubling dilution, simultaneously by step 1. in preparation Lox extracting solution be diluted to 10, 50, 100, 200 totally 4 several concentration gradients, then the reference liquid of different weaker concns and Lox extracting solution are added to respectively in the enzyme plate that 2. step get ready by each concentration 100 μ L/ hole, all establish 3 repetitions, after 37 ℃ of placement 45min-60min, according to step, 2. wash plate,
4.-1 adds soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3 monoclonal antibody reagent (L-1, L-2, L-3): soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3 monoclonal antibody reagent (L-1, L-2, L-3) are joined respectively in the enzyme plate hole that step 3.-1 added testing sample, 2. wash plate after placing 30-90min for 25 ℃～37 ℃ according to step;
4.-2 add soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3 monoclonal antibody reagent (L-1, L-2, L-3): soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3 monoclonal antibody reagent (L-1, L-2, L-3) are joined respectively in the enzyme plate hole that step 3.-2 added testing sample, 2. wash plate after placing 30-90min for 25 ℃～37 ℃ according to step;
5. develop the color and stop: by contain 20% 3,3 ', 5, the dehydrated alcohol substrate solution of 5 '-tetramethyl benzidine and pH are that 5.0 phosphoric acid salt citrate buffer solution substrate solution equal-volumes mix, join institute porose in, lucifuge is placed 15～20min, contains the Lox corresponding with added monoclonal antibody in the sample of colour developing, does not contain the Lox corresponding with added monoclonal antibody in the sample not developing the color; The 2M H that adds substrate solution volume 1/2
2sO
4stop buffer color development stopping;
6. microplate reader reading: parameters: 450nm, outside jolting 3 seconds;
7. the drafting of typical curve: be X-coordinate by the dilution log value of step 3.-2 Plays liquid, the OD value that the step of take obtains in is 7. ordinate zou, drafting serpentine typical curve;
8. utilize one section linear in the middle of serpentine typical curve, utilize the regression equation of this line segment and the OD value of testing sample serial dilution degree to calculate the unit of activity number of Lox.
The 6th technical problem solved by the invention is to provide a kind of test kit that detects soybean fat oxidase isoenzyme according to above-mentioned detection method, this test kit comprises box body, is located at the enzyme plate in box body and leaves the reagent in box body in, it is characterized in that the bottled detection reagent of depositing in box is comprised of following reagent: the Tris-CaCl that pH is 8.0
2damping fluid; Positive control reagent; Negative control reagent; Tween20-NaCl solution damping fluid; Anti-soybean fat oxidase isoenzyme Lox1, the Lox2 and the Lox3 monoclonal antibody reagent that by each secreted monoclonal antibody of the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox1, Lox2, Lox3, after horseradish enzyme labelling, are obtained respectively; The dehydrated alcohol substrate solution that contains 20% TMB; PH is 5.0 phosphoric acid salt citrate buffer solution substrate solutions; 2M H
2sO
4stop buffer; With distilled water, sigma company Soybean Lox type1-B product is diluted to the reference liquid that 800,000 unit of activity/ml obtains;
Wherein, the total protein of described positive control for extracting from common soybeans seed.
Wherein, described negative control is for entirely to lack from lipoxidase the total protein extracting soybean.
Compared to prior art, the invention has the advantages that:
1, the Soybean Lox type1-B product that utilizes Sigma company brings out Mammals as immunizing antigen and produces polyclonal antibody, after how anti-grand antibodies is to enzyme plate; Simultaneously under the prerequisite of purifying soybean Lox albumen not, utilize soybean fat oxidase near isogenic line century material first to use endoxan that the immune response of other albumen in experiment mice body in soybean masked by subtractive immunization method, again with the biased sample immunization experiment mouse that contains object soybean Lox albumen, bring out in Mice Body and produce Lox1, Lox2 and the single-minded antibody of Lox3, prepare specific hybridization oncocyte, avoided this difficult problem of protein purification;
2, by screening positive cell strain and cross detection, carry out simultaneously, reduced largely the workload of the strong monoclonal antibody specific that screens the difference beans Lox1 of the Chinese People's Anti-Japanese Military and Political College, Lox2 and Lox3;
While 3, detecting, polyclonal antibody has been incorporated on enzyme plate, does not need to seal and the coated elisa plate that spends the night in testing process, therefore saves time, easy to operate;
4, utilize the association reaction of antigen-antibody, by the existence of color developing detection enzyme and the content of enzyme, during detection, form " how anti-+ sample+monoclonal antibody " double-antibody sandwich, accurately and reliably, high specificity;
5, sample preparation is simple, once can detect three kinds of lipoxidases simultaneously, and once can detect a plurality of samples simultaneously, is applicable to breeding units, storage unit and processing quality testing department, is best suited for the screening of a large amount of kinds (being) in breeding.
Accompanying drawing explanation
Fig. 1 is 30 near isogenic line advanced clones detected result figure;
Fig. 2 is serpentine typical curve.
The culture presevation relating to:
Monoclonal antibody hybridoma cell strain (the Lox1-E of anti-soybean fat oxidase Lox1
1), for the splenocyte of mouse and the hybridoma cell strain of SP2/0 cell process cytogamy generation, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC5121, and preservation date is on July 26th, 2011;
Monoclonal antibody hybridoma cell strain (the Lox2-G of anti-soybean fat oxidase Lox2
5), for the splenocyte of mouse and the hybridoma cell strain of SP2/0 cell process cytogamy generation, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC5122, and preservation date is on July 26th, 2011;
Monoclonal antibody hybridoma cell strain (the Lox3-H of anti-soybean fat oxidase Lox3
8), for the splenocyte of mouse and the hybridoma cell strain of SP2/0 cell process cytogamy generation, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, its culture presevation is numbered: CGMCC5123, preservation date is on July 26th, 2011.
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiment, only for the object of illustration, never limit the scope of the invention.
Embodiment 1: the foundation of the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase isoenzyme
1.1 experiment materials:
The full Constitution of soybean fat oxidase " middle special No. 1 " is purchased from Institute of Crop Science, Chinese Academy of Agricultural Science;
The preservation number of U.S. Century near isogene based material national resources storehouse is respectively: Lox-1:WDD01483, Lox-3:WDD01485;
Lack Lox-2 enzyme soybean varieties " five-pointed star No. 2 ", authorization number: state examines beans 2004005;
BALB/C mice and mouse SP2/0 cell are purchased from Military Medical Science Institute's Experimental Animal Center (Beijing);
New zealand white rabbit: purchased from Hebei province's Experimental Animal Center;
96 hole polystyrene enzyme plates are purchased from Xiamen City Yun Peng development in science and technology company limited.
1.2 chemical reagent:
HAT substratum, Freund's complete adjuvant, Freund's incomplete adjuvant, Soybean Lox type1-B are Sigma company product;
Horseradish enzyme (HRP) is bought the magnificent transduction Science and Technology Ltd. from Beijing;
Other chemical reagent used is all purchased from Beijing Ding Guo biotechnology limited liability company.
1.3 test methods:
1. immunizing antigen preparation: get the soybean seeds of single Lox1 of lacking, single soybean seeds (can obtain by national genebank) that lacks No. 2 soybean seeds of Lox2 five-pointed star, single Lox-3 of lacking, with file, wear into bean powder (grinding respectively 10g), the product Soybean Lox type1-B10g(of Sigma company contains 3 kinds of soybean fat oxidases simultaneously, containing other soybean protein), the Tris-CaCl that above-mentioned materials is 8.0 with the pH of 8～15 times of volumes
2damping fluid mixes, lixiviate 1 hour, and 4 ℃ of centrifugal 10min of 12000rpm, get supernatant standby;
2. mouse immune: select 15 of the female mouse of pure lines BALB/C of the Quick off the mark in 4～6 week age, be divided into 3 groups (called after a, b, c groups), 5 every group, be numbered respectively 1,2,3,4, No. 5, weigh respectively each Mouse Weight mark (as table 1), get the 1. soybean seeds of the scarce Lox1 of list of middle preparation of step, single No. 2 soybean seeds of Lox2 five-pointed star that lack, single supernatant liquor that lacks the soybean seeds of Lox-3 is diluted to protein content 4mg/ml with physiological saline respectively, after adding equal-volume Freund's complete adjuvant to mix, take respectively subcutaneous multi-point injection first immunisation mouse, the immunizing dose of every mouse is 0.6mg, injection is single lack Lox1 soybean seeds (Lox-1) supernatant liquor be a group, injection is single lack Lox2 soybean seeds (Lox-2) supernatant liquor be b group, injection is single lack Lox3 soybean seeds (Lox-3) supernatant liquor be c group,
Table 1: each organizes Mouse Weight cartogram
3. inject endoxan solution (CY): by step 2. in the head mouse of exempting from respectively at immune 10min, 24h, 48h after, the endoxan solution (CY) that respectively mouse is carried out to the multi-point injection concentration 20mg/ml of nape portion with the amount of 90mg/kg according to table 1 small mouse body weight is subdued;
4. titration and again immunity: first immunisation after 1 week with step 1. in the Soybean Lox type1-B solution of preparation do antigen to step the mouse in 3. according to step 2. method again carry out immunity, immunizing dose is 20ug/, immunity is for the third time carried out at interval for 7 days, the 7 days same methods in interval carry out the 4th immunity, after immunity, within the 4th day, from each mouse tail, getting respectively blood 2-3 drips, centrifuging and taking supernatant, with indirect enzyme-linked immunosorbent assay, measure its tire (titration utilizes Soybean Lox type1-B to be coated with), while a, b, c3 group lacks respectively the soybean seeds (Lox-1) of Lox1 with the list of 1. middle preparation, single No. 2 soybean seeds of Lox2 five-pointed star (Lox-2) that lack, single supernatant liquor that lacks the soybean seeds (Lox-3) of Lox-3 intersects mensuration, the results are shown in Table 2, after 1 week, each mouse is carried out to the 4th immunity with Soybean Lox type1-B again according to dosage and the method for immunity for the second time, the 5th immunity carried out according to same method in 7 days in interval, after four days, get as stated above decimal tail serum, carry out titration, the results are shown in Table 3.
Table 2: each mouse tail serum titer measurement result after the 4th immunity
Table 3: each mouse tail serum titer measurement result after the 5th immunity
5. booster immunization: according to tiring with tiring and differ the principle being the bigger the better with other soybean protein with Type1-B, after the 5th immunity, No. 1 mouse of a group selection, No. 4 mouse of b group selection, No. 4 mouse of c group selection, in cytogamy, within first 3 days, by step 1-4 immunization method, adopt Soybean Lox type1-B40ug, each booster immunization 1 time;
6. cytogamy: get respectively step 5. in the splenocyte of booster immunization mouse and the SP2/0 cell of recovery mix, by known ordinary method, do cytogamy, cell after merging is added in 96 porocyte culture plates, with HAT, selects substratum to screen respectively hybridoma;
7. specificity positive cell strain screening: cytogamy is after 13 days, to the long hole that has hybridoma, adopt indirect enzyme-linked immunosorbent assay, utilize Soybean Lox type1-B to carry out positive-selecting as detectable antigens, simultaneously to a, b, c group mouse fused cell utilizes respectively the soybean seeds (Lox-1) of single Lox1 of lacking, single No. 2 soybean seeds of Lox2 five-pointed star (Lox-2) that lack, single bean powder supernatant liquor that lacks the soybean seeds (Lox-3) of Lox-3 carries out cross detection as detectable antigens, the ratio of the OD450 value of supernatant liquor and the OD450 value in blank hole be greater than 2.1 be judged as positive hole, and the hole that cross detection is negative (antibody that this hole inner cell strain secretion is described is specific antibody), hybridoma wherein can be described as positive cell strain, a, b, c group screens respectively 18, 25, 14 positive holes.
8. subclone: adopt the cell in the positive hole that limiting dilution assay screens in 7. step to carry out respectively subclone, weed out false positive cell strain after continuous three subclones, a, b, c group obtain respectively the cell strain that 1 strain is positive, respectively called after Lox1-E
1, Lox2-G
5, Lox3-H
8, being deposited in respectively China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation numbering is respectively: CGMCC5121, CGMCC5122 and CGMCC5123; In testing process, all according to the method in 7., carry out the positive and cross detection simultaneously;
Table 4:Lox1-E
1subclone situation cartogram
Table 5:Lox2-G
5subclone situation cartogram
Table 6:Lox3-H
8subclone situation cartogram
Embodiment 2: the preparation of monoclonal antibody, detection and application
2.1 detect antibody preparation
2.1.1 monoclonal antibody preparation and purification
Monoclonal antibody ascites preparation: the cell strain Lox1-E that step in embodiment 1 is obtained in 8.
1, Lox2-G
5, Lox3-H
8use respectively culturing bottle enlarged culturing, with 1 * 10
6individual cell/amount is only inoculated into through the pretreated BALB/C mice abdominal cavity of paraffin oil, and induction ascites produces, and each cell strain is inoculated respectively 5 mouse, after 7 days, can gather ascites, Lox1-E
1, Lox2-G
5, Lox3-H
8obtain respectively odd contradictive hydroperitoneum 25ml, 19ml, 23ml.
Monoclonal antibody titer of ascites is measured: with indirect ELISA method, the product Soybean Lox type1-B of Sigma company, as detectable antigens, measures Lox1-E
1, Lox2-G
5, Lox3-H
8tiring of monoclonal antibody ascites, result is tired and is respectively 1:32 ten thousand, 1:64 ten thousand, 1:32 ten thousand.
The purifying of monoclonal antibody: resulting each monoclonal antibody adopts caprylic acid-ammonium to carry out separation and purification, and step is as follows:
1. use 0.06M acetate buffer solution dilution ascites or serum 3-4 doubly, with NaOH, adjusting pH is 4.5, and writes down liquid volume;
2. under vibration, drip sad (in the ascites of every milliliter of dilution or serum, adding 25ul sad), under room temperature, stir 30min, 4 ℃, the centrifugal 30min of 10000rpm, abandons precipitation, stays supernatant.(adding sad must drop by drop adding);
3. filtering supernatant, adds the every 10 portions of supernatant liquors of PBS(of 0.1M pH7.4 to add 1 part of PBS), with NaOH, adjust pH to be 7.4,4 ℃ and place 30min;
4. grind ammonium sulfate, every milliliter of mixed solution adds 0.28g ammonium sulfate, stirs 30min, the centrifugal 20min of 10000rpm;
5. get precipitation and be dissolved in former ascites or serum 1/10 volume, with 0.01M PBS, dissolve, by the 0.01M PBS damping fluid dialysed overnight of 50 times of volumes, change liquid 2-3 time;
6. liquid is taken out from dialysis tubing, 4 ℃, the centrifugal 20min of 10000rpm, goes precipitation, is the IgG of purifying, and concentrated latter-20 ℃ save backup.
2.1.2 horseradish enzyme labelling
The step that the monoclonal antibody enzyme mark preparation of the beans Lox1 of the Chinese People's Anti-Japanese Military and Political College, Lox2 and Lox3 is detected to antibody is as follows:
1. take the HRP enzyme of 3 parts of 4mg, be dissolved in respectively in tri-distilled water, add the sodium periodate solution of recently prepared 0.1mol/k0.25ml, mix rearmounted 4 ℃, 30min, add ethanolic soln, room temperature, 30min, add respectively soybean Lox1, Lox2 and the Lox3 monoclonal antibody after 2mg purifying and make marks, mix respectively, adjust pH to 9.0,4 ℃ are spent the night;
2. add respectively sodium borohydride 0.4mg, mix, put 4 ℃ of 2h;
3. each enzyme labelled antibody mixed solution is added respectively to equal-volume saturated ammonium sulphate solution, put after 4 ℃ of 30min centrifugal, by pH7.4 phosphate buffered saline buffer dialysed overnight.
2.2 soybean fat oxidase polyclonal antibody preparations
Step prepared by soybean fat oxidase polyclonal antibody is as follows:
1. immune animal is selected and initial immunity: select the purebred new zealand white rabbit of weight between 2～3kg, Soybean Lox type1-B albumen is diluted to 8mg/ml as antigen, after isopyknic Freund's complete adjuvant mixing and emulsifying, take the subcutaneous multi-point injection immune rabbit in back, every rabbit immunizing dose is 1.6mg;
2. immunity again: immunity is again carried out according to step after utilizing afterwards the Soybean Lox type1-B albumen equal-volume mixing that incomplete Fu Shi adjuvant and concentration are 8mg/ml for 14 days in interval 1., so carries out immunity for the third time;
3. titration: for the third time after immunity the 11st day from above-mentioned steps 2. immune White Rabbit auricular vein get blood examination and survey it and tire, if tired while being less than 1:10000, to step 2. in White Rabbit again according to 2. immunity of step, so repeatedly immunity until survey and tire higher than 1:10000;
4. the preparation of polyclonal antibody: the White Rabbit of 3. tiring the highest to step after 3 days adopts the blood sampling of carotid artery depletion method, separation of serum, adopt indirect enzyme-linked immunosorbent assay to measure intersecting of other albumen in separation of serum and Lox1, Lox2, Lox3 and soybean, prove this serum and Lox1, Lox2, Lox3 all have intersect and with soybean in other albumen without obtaining polyclonal antibody after intersecting.
2.3 qualitative detection lacking lipoxygenases
Experiment material: 30 of near isogenic line advanced clones
The disappearance type and the content that detect soybean seeds central Lox1, Lox2, these 3 kinds of lipoxidases of Lox3 comprise following operation steps:
1. the preparation of Lox extracting solution in soybean to be measured: by the seed of soybean to be measured, the full Constitution of soybean fat oxidase " middle special No. 1 " seed (negative control, be labeled as A), the common soybeans seed (positive control that contains three kinds of Lox, be labeled as B) and 30 parts of detected materials respectively from a side (not destroying hilum) mill bean powder, about every part of mill 0.05g, add the Tris-CaCl of the pH8.0 of 0.5ml
2damping fluid, mixes rear centrifuging and taking supernatant standby;
2. wash plate: with Tween20-NaCl damping fluid, 96 coated hole polystyrene enzyme plates of polyclonal antibody are washed 3～4 times repeatedly, each 2～3min, pats dry, and object washes away the polyclonal antibody not being attached on plank;
3. application of sample: by step 1. in the Lox extracting solution of preparation by being added to shown in table 7 in the enzyme plate that 2. step get ready, every hole 100 μ L, each sample adds 3 holes, wherein makes respectively feminine gender and positive control with common soybeans middle special No. 1, after 37 ℃ of placement 30min, according to step, 2. washes plate;
Table 7 is for each reacting hole position signal table of enzyme plate of qualitative detection
| ? | L-1 | L-2 | L-3 | L-1 | L-2 | L-3 | L-1 | L-2 | L-3 | L-1 | L-2 | L-3 |
| A | A | A | A | B | B | B | No. 1 | No. 1 | No. 1 | No. 2 | No. 2 | No. 2 |
| B | No. 3 | No. 3 | No. 3 | No. 4 | No. 4 | No. 4 | No. 5 | No. 5 | No. 5 | No. 6 | No. 6 | No. 6 |
| C | No. 7 | No. 7 | No. 7 | No. 8 | No. 8 | No. 8 | No. 9 | No. 9 | No. 9 | No. 10 | No. 10 | No. 10 |
| D | No. 11 | No. 11 | No. 11 | No. 12 | No. 12 | No. 12 | No. 13 | No. 13 | No. 13 | No. 14 | No. 14 | No. 14 |
| E | No. 15 | No. 15 | No. 15 | No. 16 | No. 16 | No. 16 | No. 17 | No. 17 | No. 17 | No. 18 | No. 18 | No. 18 |
| F | No. 19 | No. 19 | No. 19 | No. 20 | No. 20 | No. 20 | No. 21 | No. 21 | No. 21 | No. 22 | No. 22 | No. 22 |
| G | No. 23 | No. 23 | No. 23 | No. 24 | No. 24 | No. 24 | No. 25 | No. 25 | No. 25 | No. 26 | No. 26 | No. 26 |
| H | No. 27 | No. 27 | No. 27 | No. 28 | No. 28 | No. 28 | No. 29 | No. 29 | No. 29 | No. 30 | No. 30 | No. 30 |
4. the enzyme labelled antibody reagent (L-1, L-2, L-3) that adds soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3: the enzyme labelled antibody reagent of soybean fat oxidase isoenzyme Lox1, Lox2 and Lox3 (L-1, L-2, L-3) has 3. been added in the enzyme plate hole of testing sample according to joining respectively step shown in table 7 by every hole 100 μ L, 2. washed plate according to step after 37 ℃ of placement 30min;
5. develop the color and stop: by contain 20% 3,3 ', 5, the dehydrated alcohol substrate solution of 5 '-tetramethyl benzidine and pH are that 5.0 phosphoric acid salt citrate buffer solution substrate solution equal-volumes mix, by every hole 100 μ L join institute porose in, lucifuge is placed 15～20min, contains the Lox corresponding with added monoclonal antibody in the sample of colour developing, in the sample not developing the color, do not contain the Lox corresponding with added monoclonal antibody, result as shown in Figure 1.
That is: single material that lacks Lox1 is: No. 19, single material that lacks Lox2 is: No. 15, single material that lacks Lox3 is respectively: 1,4,7,8,18,23, No. 25, what lack Lox1 and Lox2 is No. 13 simultaneously, what lack Lox2 and Lox3 is No. 16 simultaneously, what 3 kinds of Lox lacked simultaneously is No. 3, and other material is the material that does not lack Lox.
The content of Lox1 in 2.4 detection by quantitative " Jidou15 "
1. the preparation of Lox1 extracting solution: use file to get 0.05g bean powder from 1 " Jidou15 " seed mill, be positioned in 1.5ml centrifuge tube, with 1. preparing Lox1 extracting solution in embodiment 2.3;
2. wash plate: with Tween20-NaCl damping fluid, 96 coated hole polystyrene enzyme plates of polyclonal antibody are washed 3～4 times repeatedly, each 2～3min, pats dry, and object washes away the polyclonal antibody not being attached on plank;
3. application of sample is dissolved in the soybean type-B of sigma company of 5mg in 10ml Tween20-NaCl, be made into the standardized solution of 300,000 unit of activity/L, again with Tween20-NaCl damping fluid by 10,30,90,270,810,2430,7290,21870,65610,196830,590490,0 totally 12 concentration carry out doubling dilution, the Lox extracting solution dilution 10,50,100,200 simultaneously 1. step being prepared is totally 4 concentration, application of sample shown according to the form below 8, all establish 3 repetitions, after 37 ℃ of placement 45min, according to step, 2. wash plate;
Table 8 is for each reacting hole position signal table of enzyme plate of detection by quantitative
4. add L-1: get the L-1 after 6ml dilutes 2000 times, by 100 μ L/ holes, be added in enzyme plate, after 37 ℃ of placement 30min, according to step, 2. operate and wash plate;
5. develop the color and stop: by the dehydrated alcohol substrate solution that contains 20% TMB and pH, be that 5.0 phosphoric acid salt citrate buffer solution substrate solution equal-volumes mix, by every hole 100 μ L join institute porose in, lucifuge is placed 15～20min,
6. microplate reader reading, wavelength is selected 450nm;
7. the drafting of typical curve: by step 3. the dilution log value of Plays liquid be X-coordinate, the OD value that the step of take obtains in is 6. ordinate zou, drafting serpentine typical curve, as shown in Figure 2;
8. utilize one section linear in the middle of serpentine typical curve, utilize the regression equation of this line segment and the OD value of testing sample serial dilution degree to calculate the content of Lox, the average OD under each weaker concn of standardized solution
450value table, as shown in table 9.
Average OD under each weaker concn of table 9 standardized solution
450value table
According to the average OD under each weaker concn of the standardized solution shown in table 9
450value, drawing standard curve, as shown in Figure 2.
Linear between the 4th point and the 6th point (i.e. 270 times and 2430 times of dilutions), linear equation is: y=-1.26x-3.124(0.584 < y < 1.563) as seen from Figure 2
The OD of 5 times of dilutions in the middle of detected result
450=0.606 meets this equation, and as calculated, in " yellow No. 13 of Ji ", the content of Lox1 is 7500 unit of activity/g.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and in the spirit and scope that limit, can carry out many changes to it in the claims in the present invention, revise, and even equivalence change, but all will fall within the scope of protection of the present invention.
Claims (4)
1. the monoclonal antibody hybridoma cell strain of anti-soybean fat oxidase Lox3, it is characterized in that splenocyte and SP2/0 cell that described monoclonal antibody hybridoma cell strain is mouse produce through cytogamy, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its culture presevation is numbered: CGMCC5123.
2. the monoclonal antibody of being secreted by monoclonal antibody hybridoma cell strain claimed in claim 1.
3. the application of monoclonal antibody claimed in claim 2 in detecting soybean fat oxidase Lox3.
4. monoclonal antibody claimed in claim 2 detects the application in soybean fat oxidase Lox3 test kit in preparation.
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