CN101445558B - Monoclonal antibody of glycinin and application thereof - Google Patents
Monoclonal antibody of glycinin and application thereof Download PDFInfo
- Publication number
- CN101445558B CN101445558B CN2008102410058A CN200810241005A CN101445558B CN 101445558 B CN101445558 B CN 101445558B CN 2008102410058 A CN2008102410058 A CN 2008102410058A CN 200810241005 A CN200810241005 A CN 200810241005A CN 101445558 B CN101445558 B CN 101445558B
- Authority
- CN
- China
- Prior art keywords
- glycinin
- monoclonal antibody
- antibody
- cgmcc
- legumin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a monoclonal antibody of glycinin and the application thereof. Monoclonal antibody of glycinin provided by the invention is made of anti-glycinin monoclonal antibody cell line 4B2 with preserving number being CGMCC No.2791. When being used for detecting the glycinin, the glycerin monoclonal antibody of the invention has the advantages that the defects, such as no reference conducted between different laboratories, low sensitivity and accuracy, and the repeated preparation for polyvalent antibody in an immune manner, existing in the traditional methods, such as SDS-PAGE and polyvalent antibody indirect ELISA; the detection accuracy is close to that obtained by adopting reversed-phase hplc method (RP-HPLC); the cost is very low; the requirement for detecting the content of glycinin in soybeans, bean pulp, and bean products in a high throughput manner by the feed industry, the food industry, and relevant enterprises; and the broad application prospect of quantitatively detecting antigenic substances of bean products and evaluating the hypersensitivity-inducing ability is possessed.
Description
Technical field
The present invention relates to a kind of monoclonal antibody and application thereof of glycinin.
Background technology
Soybean has widespread use as humans and animals fine vegetable-protein and oil source.Soybean and bean product are widely used in food service industry, and corn-soybean meal diet is widely used in Production of Livestock and Poultry as main animal-feed.But glycinin is abundant as content, thermostability is strong, have stronger immunogenic soybean antigen albumen, often cause allergic reaction, often show as infantile diarrhea at medical field, and the grownup of the children of 5-6% and 2-4% shows clinical symptom to soybean and bean product, often show as diarrhoea, serious with tremble, symptom such as edema of throat and acute asthma; In the herding industry, can cause the weaning anaphylaxis of young animals such as piglet and pregnant sow, calf, rat of soybean antigen albumen often shows as diarrhoea, growth performance descends until death.
Developed multiple complete processing at present and handled soybean, dregs of beans and bean product, be weaker than raw soybean as the multinomial antigenic activity of soya productss such as dregs of beans, expanded soybean, fermented bean dregs, soybean protein concentrate and soybean protein isolate that studies confirm that.Yet glycinin is the antigen protein of thermostability, and direct heating can not thoroughly destroy its antigenic activity.For example, the sphaeroprotein residual quantity that has antigenic activity in the process soya products of heat treated is still up to 18%, and this amount is enough to cause baby's anaphylaxis.
So the detection of different complete processings being handled glycinin content in front and back soybean, bean product, the bean-dregs feed has important significance for theories and using value.Existing quantitative detecting method is mainly measured and efficient liquid phase chromatographic analysis based on antiserum(antisera).Still there is certain defective in these methods from the immunology angle, the former polyvalent antibody need repeat preparation, can not reference between the different experiments chamber, sensitivity and accuracy be low; The latter then can't embody immunoreactivity, and specialized equipment loaded down with trivial details owing to the pre-treatment process and that needs are expensive is difficult to be widely used in the scene detection of soya products.
Summary of the invention
An object of the present invention is to provide a kind of can be used for the detecting monoclonal antibody of glycinin and the hybridoma of this antibody of secretion.
The monoclonal antibody of secretion glycinin provided by the present invention is the legumin cell strain of monoclonal antibody 4B2 of the Chinese People's Anti-Japanese Military and Political College generation of CGMCC No.2791 by preserving number.This monoclonal antibody is the mouse IgG that comprises the κ light chain
2Antibody, its affinity costant (Ka) is 3.3 * 10
8L/mol.
The legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 05th, 2008 and (is called for short CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preservation registration number is CGMCC No.2791.
Another object of the present invention provides the test kit of the detection glycinin that contains said monoclonal antibody, and described test kit is an enzyme-linked immunologic detecting kit.
For more convenient on-site supervision and great amount of samples examination, in the test kit of the detection glycinin that contains said monoclonal antibody provided by the present invention, also contain other enzyme linked immunosorbent detection reagent, as ELIAS secondary antibody, glycinin standard solution, colour developing liquid, stop buffer, washings etc.
With the monoclonal antibody of above-mentioned glycinin and the solid phase carrier immune affinity sorbent that obtains of coupling mutually, be that the immune affinity chromatographic column of filler and the test kit that contains this immune affinity sorbent and immune affinity chromatographic column all belong to protection scope of the present invention with this immune affinity sorbent.
Wherein, described solid phase carrier can be affinity chromatography carriers such as Mierocrystalline cellulose, dextrane gel, polyacrylamide gel, sintered glass, sepharose or ultragel ACA22.
The glycinin monoclonal antibody of the present invention preparation for fundamental research, screening varieties and transformation, the clinical diagnosis of glycinin provides favourable instrument, can be used for furtheing investigate the biological effect of glycinin.Particularly can be applicable to:
(1) detects glycinin, as the content of glycinin in feedstuff industry, food service industry high-throughput rapid detection soybean, dregs of beans, bean product, the bean-dregs feed;
(2), carry out the screening of soybean germplasm resource, kind transformation with the instrument of this antibody as the research soybean antigen;
(3) use this Antibody Preparation immune affinity column, come the separation and purification glycinin;
(4) use this antibody and carry out animal experiment study;
(5) the glycinin monoclonal antibody is carried out mark (as carrying out mark), be used for clinical diagnosis or auxiliary diagnosis patient the soybean food anaphylaxis with FITC;
(6) use this antibody and carry out of the distribution of immunohistochemical methods detection glycinin at organ-tissue, various body fluid;
(7) use the antagonist of this antibody, can carry out pharmaceutical research, be used for the treatment of soybean antigen albumen anaphylactic disease as glycinin.
Detect glycinin with glycinin monoclonal antibody of the present invention, overcome traditional SDS-PAGE, can not reference between the different experiments chamber that polyvalent antibody indirect ELISA detection method exists, sensitivity and accuracy are low, need to repeat the shortcoming of immunity preparation polyvalent antibody, its detection accuracy is near reversed phase high efficiency liquid phase method (RP-HPLC), and cost is very low, can satisfy feedstuff industry, food service industry relevant enterprise unit high-throughput rapid detection soybean, dregs of beans, the needs of glycinin content in the bean product have broad application prospects with causing in hypersensitive assessment in soya products antigenic substance detection by quantitative.
Description of drawings
Fig. 1 analyzes the glycinin sample of purifying for SDS-PAGE.Band a among the figure: blank; Band b: the glycinin crude samples of extraction; Band c: the glycinin sample of purifying.
The curve of tiring of the monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of Fig. 2 Chinese People's Anti-Japanese Military and Political College is produced.The mean value (n=8) of each some representative 8 replications in elisa assay among the figure.The concentration of envelope antigen glycinin is 50.0 μ g/mL, and the initial concentration of monoclonal antibody is 1.0mg/mL.(X-coordinate is meant 10 n power, such as 4 correspondences is exactly 10
4, i.e. 10000 times of this some representative dilutions).
The monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of Fig. 3 Chinese People's Anti-Japanese Military and Political College is produced and the immunoblotting assay of soybean and goods thereof.Band a among the figure: the glycinin crude samples of extraction; Band b: the glycinin sample of purifying.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.Used reagent among the following embodiment if no special instructions, all obtains from commercial channels.
1, the extraction of glycinin and purifying
The method preparation of adopting salt fractionation to combine with gel-filtration.Concrete steps are as follows:
1.1. defibrination behind the soybean peeling filters back extracted with diethyl ether degreasing, removes residual ether through rotary evaporation, makes defatted soyflour.
1.2. at ambient temperature, the Tris-HCl of adding 0.03M in defatted soyflour (pH8.0, and the 10mM beta-mercaptoethanol (available from: Shanghai Aladdin, article No.: 1093795)) damping fluid (1: 20), dissolving, 10, the centrifugal 30min of 000rpm/min collects supernatant liquor.
1.3. regulate pH to 6.4 with HCl, 10, the centrifugal 30min of 000rpm/min (4 ℃), collecting precipitation (adds the 0.01mol/L phosphoric acid buffer (PBS) of 10mmol/L beta-mercaptoethanol, pH8.0) (0.01mol/L phosphoric acid buffer (PBS): 8.00gNaCl, 0.20g KCl, 0.20g KH with phosphate buffered saline buffer
2PO
4, 1.15gNa
2HPO
412H
2O, adding distil water be to 1000mL, and transferring PH with the NaOH of 1mol/L is 8.0).Dissolving, centrifugal collection supernatant liquor obtains the glycinin crude extract.
1.4. add solid (NH
4)
2SO
4, make (NH
4)
2SO
4After saturation ratio reaches 51%, 4 ℃ of standing over night, 15,4 ℃ of centrifugal 30min collecting precipitations of 000rpm/min, resolution of precipitate is in the phosphate buffered saline buffer of pH7.6.Add solid (NH
4)
2SO
4, make (NH
4)
2SO
4After saturation ratio reaches 90%, 4 ℃ of standing over night, with 15, the centrifugal 30min of 000rpm/min (4 ℃), precipitation is dissolved in the phosphate buffered saline buffer of pH7.6.
(100cm * 2.6cm), fully go up sample after the balance and carry out gel-filtration, with the phosphate buffered saline buffer wash-out of pH7.6, nucleic acid-protein detector 280nm detects, and collects eluant component respectively 1.5. sepharose Sepharose 6B adorns post after vacuum outgas.The proteic elutriant of collecting that contains is passed through DEAE-52 chromatography column (30cm * 3.3cm), collect the albumen elutriant again.With elutriant in 4 ℃ to distill water dialysis desalination, lyophilize.
1.6. purity of protein is identified and molecular weight determination adopts the SDS-PAGE electrophoresis method to carry out, the separating gel concentration of adding SDS is 12%, and concentrated gum concentration is 5%.With the lower molecular weight standard protein is that standard is carried out electrophoresis, and deposition condition is constant voltage 100V.Electrophoresis finishes the back with more than the Coomassie brilliant blue staining fluid dyeing 6h, and decolouring is observed, and the result as shown in Figure 1.
1.7. utilize the BCA protein detection kit to detect proteic concentration, determination step is undertaken by operation instructions.
2, the foundation of hybridoma cell line
2.1. animal immune
The glycinin solution of an amount of purifying is added in the 0.5mL physiological saline, be made into 5mg/mL concentration, make the immunogen emulsifying agent, immunity female Balb/c mouse in 6 ages in week after adding isopyknic complete Freund's adjuvant (available from Sigma, catalog number (Cat.No.) F5506-10).Row immunity for the second time after 4 weeks, two Zhou Houhang immunity for the third time again, all use is made the immunogen emulsifying agent after adding isopyknic incomplete Freund's adjuvant (available from Sigma, catalog number (Cat.No.) F5881-10).Measured glycinin antibody titer in the mice serum at immune back the 10th day for the third time with indirect elisa method, the high person that tires carries out next step cytogamy.
2.2. cytogamy
Aseptic drawing materials through the Balb/c mouse spleen of step 2.1. immunity made splenocyte suspension.Draw respectively and contain 1 * 10
8Individual splenocyte and 2 * 10
7The suspension of individual myeloma cell sp2/0 moves in the 50mL centrifuge tube.Add incomplete nutrient solution, making the enchylema cumulative volume is 30mL.Fully behind the mixing,, abandon supernatant liquor in the centrifugal 7min of 1000 * g.Flick the pipe end, loose sedimentation cell.The centrifuge tube bottom is immersed in 37 ℃ of warm water, evenly rotated, with the centrifuge tube of 1mL 50%PEG solution, about 60 seconds application of sample time along centrifugal tube wall application of sample rotation.Immediately cell suspending liquid is all sucked suction pipe, leave standstill 30 seconds after, again it is blown in the centrifuge tube.In 5min, add the incomplete nutrient solution of 25mL with dilution PEG, make PEG lose the short effect of melting.In the centrifugal 7min of 800 * g, abandon supernatant liquor.Add 10mL HAT nutrient solution, suspend and the mixing sedimentation cell.The cell suspending liquid adding has been covered with in 96 well culture plates of feeder cell each hole 0.1mL.Then culture plate is placed 37 ℃ and 5%CO
2Incubator in cultivate.
Cell suspension after merging in the HAT nutrient solution, is placed 37 ℃ and 5%CO
2Incubator in cultivate.Changed nutrient solution once in every 2-3 days.Use the HAT nutrient solution in fusion in back 7 days; Used the HT nutrient solution in 7-14 days instead; Then used later common complete culture solution on the 14th day.
2.3. indirect immunoperoxidase connection adsorption experiment (ELISA) screening positive hybridoma cell
After cytogamy the 7th day, change liquid at every turn and collect Hybridoma Cell Culture liquid, adopt indirect ELISA method the nutrient solution in each cell cultures hole to be carried out the screening of positive hybridoma cell strain then.Concrete operations are as follows:
(1) bag quilt: be cushioned liquid ((carbonic acid buffer of 0.85mol/L, pH9.6): accurately take by weighing 1.59g Na with bag
2CO
3With 2.93g NaHCO
3, it is miscible in 1000mL distilled water) with purifying glycinin solution dilution to best effort concentration (2.5 μ g/mL), application of sample 100 μ L in every hole put wet box in 37 ℃ of incubations 1 hour.
(2) sealing: discard the liquid in the enzyme plate hole.Each hole adds confining liquid (be cushioned the liquid preparation with bag, add 1% gelatin) 150 μ L, puts in the box that wets 37 ℃ of incubations after 1 hour, with lavation buffer solution (the 0.01mol/L PBS (pH7.4) that contains 0.05%Tween-20; 0.01mol/L (PBS, prescription pH7.4) are 8.00g NaCl to phosphoric acid buffer, 0.20g KCl, 0.20g KH
2PO
4, 1.15g Na
2HPO
412H
2O; Adding distil water is to 1000mL) wash 3 times, each 90s (being called for short washing, down together) pats dry then.
(3) add testing sample: the Hybridoma Cell Culture supernatant liquor is added enzyme plate after with suitable initial concentration doubling dilution with antibody diluent (0.01mol/LPBS (pH7.4) that contains 0.01%Tween-20,0.1% gelatin), each hole 100 μ L, each concentration gradient is established 3 repetitions, establishes blank, negative control and positive control simultaneously.Behind 37 ℃ of incubation 1h, washing pats dry.
(4) add ELIAS secondary antibody (anti-antibody): with antibody diluent with ELIAS secondary antibody (sheep anti-mouse igg/HRP) is diluted to working concentration (1: 5000), adds enzyme plate, each hole 100 μ L, place 37 ℃ of incubation 1h after, washing pats dry.
(5) add substrate solution: add freshly prepared OPD substrate to each enzyme mark hole and use liquid (OPD substrate use liquid: 40mg OPD is dissolved in the 100mL substrate buffer solution, adds 30 μ L 30%H again
2O
2Substrate buffer solution ((phosphoric acid-citrate buffer solution of pH5.0): the 0.2mol/L Sodium phosphate dibasic (28.4g/L) of 25.7mL and the 0.1mol/L citric acid (19.2g/L) of 24.3mL are dissolved in 50mL distilled water) 100 μ L, 37 ℃ of incubation 15-30mm.
(6) termination reaction: each hole adds stop buffer ((2mol/L H
2SO
4): the 21.7mL vitriol oil (98%) is dissolved in the 156.6mL distilled water) 50 μ L, the substrate color reaction stopped.
(7) result of determination: the light absorption value (OD that under the 492nm wavelength, measures solution in each hole with microplate reader
492), if treat gaging hole OD
492More than or equal to 2.1 times of negative control hole, promptly think positive value, thereby draw possible positive hybridoma cell clone.
2.4. the mono-clonalization of positive hybridoma cell
Adopt limiting dilution assay that the positive cell strain of screening is cloned, concrete steps are as follows: prepare feeder cell (the abdominal cavity mononuclear macrophage of Balb/c mouse) the day before yesterday in the clone.After blowing and beating evenly repeatedly with sample injector hybridoma to be cloned, go in the 24 porocyte culture plates, and carry out cell counting.According to count results, pair cell suspension is done suitable dilution.Get 250 viable cell and be suspended in (contain 5 cells in average every 0.1mL solution this moment) in 4.6mL (final volume) nutrient solution, inoculate 96 well culture plates, each hole 0.1mL, totally 36 holes.The 4mL nutrient solution is joined (contain 1 cell in average every 0.1mL solution this moment) in the remaining 1.0mL cell solution, this enchylema is seeded to next 36 holes, each hole 0.1mL.In remaining 1.4mL cell suspending liquid, add nutrient solution 1.4mL (contain 0.5 cell in average every 0.1mL solution this moment).Behind the mixing, it is inoculated in remaining 24 holes, each hole 0.1mL.Culture plate is placed 37 ℃ and 5%CO
2Cultivate in the incubator.
Change liquid and detection in good time.When having porous positive, should get the mono-clonal hole as far as possible and carry out time cloning again, all positive until the nutrient solution in all cells hole.Obtain the legumin cell strain of monoclonal antibody 4B2 of the stably excreting Chinese People's Anti-Japanese Military and Political College at last, this cell strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 05th, 2008 and (is called for short CGMCC, the address is: Da Tun road, Chaoyang District, BeiJing, China city), preservation registration number is CGMCC No.2791.
3, extracorporeal culture-ing prepares monoclonal antibody
The legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College that has set up is carried out enlarged culturing, and used substratum places 37 ℃ and 5%CO for the DMEM substratum adds 20% foetal calf serum
2Cultivate in the incubator, treat that cell concn is greater than 10
5Stop to change liquid during/ml, continue to hatch to cell all dead.Collect culture supernatant, 1500-2000 * g, centrifugal 10 minutes, supernatant liquor contained high-caliber monoclonal antibody, and-20 ℃ of preservations are standby.
4, induce the ascites legal system in the body and be equipped with monoclonal antibody
A large amount of preparations of antibody are adopted in the body and are induced method.Whiteruss (0.5mL/ only) is injected healthy Balb/c female mice intraperitoneal, and 1-2 is standby after week.The legumin cell strain of monoclonal antibody 4B2CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College of enlarged culturing is blown off, and collecting cell behind the centrifugal 5min of 1000 * g is abandoned supernatant.Too many or too much for use full nutrient solution with cell suspension, and mixing is adjusted to 10 with cell concn
6Individual/mL.Injection 1mL/ above-mentioned hybridoma only in the mouse peritoneal that carries out Treating Cuttings with Paraffin Wax.Collect ascites after 7-9 days,, abandon lipid layer and cellular layer in the centrifugal 10min of 3000 * g, clear layer in the middle of collecting, place-70 ℃ frozen.
5, the purifying of monoclonal antibody
5.1. pre-treatment (silicon-dioxide absorption method)
Get the supernatant liquor of step 3 acquisition or the mouse ascites that step 4 obtains, with isopyknic barbitol buffer solution dilution.Add SiO 2 powder, shake 30min under the room temperature frequently.In the centrifugal 20min of 2000 * g, promptly get clarifying liquid.
5.2. it is sad-the ammonium sulfate precipitation method IgG purification
The 0.06mol/L acetate buffer (pH5.0) that in the pretreated liquid of portion process step 5.1., adds 2 times of volumes.With 0.1mol/L HCl adjust pH to 4.8.Under room temperature, stir, and in 30min, dropwise add sad (it is sad that the pretreated liquid of process step 5.1. before the every 1mL dilution adds 33 μ L).4 ℃ leave standstill 2h after, in the centrifugal 30min of 15000 * g.Abandon precipitation, supernatant liquor filters through sand core funnel.In solution, add 0.1mol/L PBS, with 1mol/L NaOH adjust pH to 7.4 through 1/10 volume of the pretreated liquid of step 1.Place 4 ℃ of ice baths, in 30min, add (the NH of 0.277g/mL
4)
2SO
4, make its saturation ratio reach 45%.After leaving standstill more than the 1h, in the centrifugal 30min of 10000 * g.Abandon supernatant, with precipitation be dissolved in PBS (contain 137mmol/L NaCl, 2.6mmol/L KCl and 0.2mmol/L EDTA, pH7.4) in.4 ℃ of dialysed overnight in PBS.Behind the centrifugal 30min of 10000 * g, remove insoluble sediment, clear liquor is elementary pure antibody.
5.3. affinitive layer purification IgG
Being further purified of antibody adopts HiTrap Protein G HP affinity column to carry out.Concrete operations are as follows: with binding buffer liquid (20mmol/L phosphoric acid salt, pH7.0) the balance chromatography column of 10 times of column volumes (about 10mL).With diameter is that the strainer filtered sample of 0.45 μ m is removed insolubles, mixes by 1: 1 (V/V) with binding buffer liquid then.Use syringe sampling again, clean with 10 times of column volumes (about 10mL) binding buffer liquid subsequently, to remove not in conjunction with foreign protein.At last with elution buffer (0.1mol/L glycine-hydrochloric acid, pH2.7) elution samples of 2-5 times of column volume.Collect elutriant (collect the Tris-HCl neutralizer that test tube is added with 1mol/L pH9.0 in advance, its consumption is that 100 μ L/mL collect liquid).Fully dialysis in 5mmol/L PBS concentrates standby then.
6, the CHARACTERISTICS IDENTIFICATION of monoclonal antibody
6.1. the type of antibody and hypotype
Get the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of Chinese People's Anti-Japanese Military and Political College nutrient solution, in the centrifugal 8min of 2000 * g, to remove cell and fragment thereof.Measure the hypotype of type, hypotype and the light chain of antibody with the pure monoclonal antibody parting kit of immunity I (ImmunoPureMonoclonal Antibody Isotyping Kit I).According to the parting kit analysis, the monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College produces belongs to the IgG that contains the k light chain
2
6.2. the content of antibody
Collect the nutrient solution of the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College, adopt the antibody concentration in the sandwich ELISA method mensuration nutrient solution then, with purifying rabbit anti-mouse igg coated elisa plate, the washing back adds the monoclonal antibody that rabbit anti-mouse igg-the HRP identification legumin cell strain of monoclonal antibody 4B2CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced of suitable concentration.Different concns with mouse IgG is an X-coordinate, with the OD of correspondence
492Value is ordinate zou, drawing standard curve.According to the OD that detects each strain of hybridoma nutrient solution
492Be worth, calculate the accurate concentration of antibody.
6.3. tiring of antibody
Glycinin (50.0 μ g/mL) coated elisa plate with step 1.After the monoclonal antibody (initial concentration of monoclonal antibody is 1.0mg/mL) of purifying carried out doubling dilution, measure tiring of antibody, with the negative contrast of culture supernatant of the SP2/0 cell that merges with indirect ELISA method.Fig. 2 is the graphic representation of tiring of antibody, with OD
492Value is that 1.0 corresponding antibody titers are 2.7 * 10
4(mean value of each some representative 8 replications in elisa assay among the figure).
6.4. the detection of monoclonal antibody stability
The recovery hybridoma is collected the cell conditioned medium of different passage numbers, and is detected it with indirect ELISA method and tire.The result shows that the different generations of the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College can be stablized and produces monoclonal antibody (with embodiment 1, the described method of step 6.3 detects, and the antibody titer in this cell strain 8 generations of the 1st generation to all is higher than 1 * 10
4).
6.5. the mensuration of affinity of antibody (noncompetitive ELISA)
The affinity costant of antibody adopts noncompetitive ELISA method, and schedule of operation is except the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College nutrient solution of the concentration known of adding serial dilution, and other are with step 2.3.
Different concns with testing sample is an X-coordinate, with its corresponding OD
492Value is ordinate zou, draws three and measures curve.Be tending towards smooth OD with each curve top
492Value is 100%, calculates OD
492Value is concentration [Ab] t of 50% o'clock antibody, and every part of testing sample can get [Ab] t, [Ab] ' t, [Ab] like this, and " three values of t are then according to Xu Zhikai (Xu Zhikai chief editor, 1991, practical monoclonal antibody technique.Xi'an: Shaanxi science tech publishing house.Pp 7-145) method of describing is calculated, the affinity costant (K of the monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced
a) be 3.3 * 10
8L/mol.
6.6. the atopic of monoclonal antibody
(1) antibody and analogue cross reaction
With the cross reaction between competitive ELISA mensuration glycinin and its structure or the functional analogue.After the monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced is done the proper concn dilution, with equal-volume respectively with the glycinin of serial doubling dilution, β-companion poly-sphaeroprotein, trypsin ihhibitor and lectin mixing after, room temperature reaction 15min.Add to then and wrapped in the processed enzyme plate.The same 2.3. of all the other steps.Wherein, envelope antigen (glycinin) concentration that is used for elisa assay is 50.0 μ g/mL, and the monoclonal anti bulk concentration that glycinin cell strain of monoclonal antibody 4B2 CGMCC No.2791 is produced is 50ng/mL.According to OD
492Value drawing standard curve is determined IC
50(ng/mL) (IC
50Respectively compete substrate concentration, each IC in the table 1 during for inhibition antigen-antibody bonded 50%
50Represent the mean value of 12 replications).The cross reacting rate that calculates each analogue according to following formula (is the IC of glycinin
50With each competition thing IC
50The ratio.Cross reacting rate CR (%)=(IC of glycinin
50The IC of certain competition thing of)/(
50) * 100%.As shown in table 1.
The numerical value of secondary series lastrow is IC in the table 1
50, represent that with mean+SD unit is ng/mL; The numerical value of next line is cross reacting rate, and unit is %.
The cross reaction of table 1. antibody and glycinin and analog thereof or related substances
| The competition thing | IC 50, mean+SD, and ng/mL (cross reacting rate CR, %) |
| Glycinin | 12.2±3.5 (100.0) |
| β-companion gathers sphaeroprotein | 3597±10.8 (0.3) |
| Trypsin ihhibitor | (＜0.05) |
| Lectin | (＜0.05) |
(2) detect monoclonal antibody and glycinin association reaction with Western Blot immunoblot experiment
The glycinin of thick leach protein of soybean and purifying is carried out the 10%SDS-PAGE electrophoresis, in Bio-Rad electrotransfer system, the gel protein band is transferred on the nitrocellulose filter according to a conventional method, spend the night 4 ℃ of sealings with 5% skim-milk, add the monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced.Room temperature reaction 1h washs three times.The sheep anti-mouse igg that adds alkali phosphatase enzyme mark subsequently, room temperature reaction 1h washs three times.Add the colour developing of NBT/BCIP substrate at last.TBST is all used in washing, each 10min.The result as shown in Figure 3, the result shows that monoclonal antibody and glycinin that the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced have stronger specific reaction, band a: the glycinin crude samples of extraction; Band b: the glycinin sample of purifying.
The result of ELISA and Western Blot proves: monoclonal anti physical efficiency and glycinin generation intensive association reaction that the legumin cell strain of monoclonal antibody 4B2CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced, therefore the immunology detection and the protein quantification that can be applied to, and other related application research.
Embodiment 2: the double antibodies sandwich ELISA detection method of glycinin
The monoclonal antibody that is produced with the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College detects glycinin and can comprise following reagent:
1) monoclonal antibody that produced of Chinese People's Anti-Japanese Military and Political College legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 is carried out purifying by embodiment 1 step 5;
2) polyclonal antibody of Chinese People's Anti-Japanese Military and Political College legumin.Preparation, the purification process that should resist as follows more:
A. many anti-preparations
The glycinin of getting embodiment 1 preparation is made into 5mg/mL with physiological saline, mixes with the equivalent freund's adjuvant, utilizes vortex mixer to make its emulsification complete.Choose the Healthy female new zealand white rabbit, first immunisation is the inboard epithelium of foot, and injection Freund's complete adjuvant emulsification antigen selects two sites, every some 0.5mL; Head exempts from back 14 days booster immunizations, back picked at random 4 points, sterilization back subcutaneous injection Freund emulsification antigen, 0.5mL/ point; Later on once, continuous three booster immunizations every the 10d booster immunization.10d behind the last booster immunization selects vein blood vessel clearly on ear, injection suprarenin 0.1mL/ only, cross 0.5h after, the antigen of purifying again toward the vein direct injection.Wait to tire reach requirement after, rabbit is carried out precaval vein blood sampling, separation of serum ,-20 ℃ of preservations are standby.
B. many anti-purifying
3) sheep anti-mouse igg/HRP ELIAS secondary antibody;
4) glycinin standard substance (can be prepared purifying) according to the method for embodiment 1 step 1;
5) bag is cushioned liquid (forming with embodiment 1 step 2.3. (1));
6) 1% bovine serum albumin confining liquid (composition) with embodiment 1 step 2.3. (2);
7) lavation buffer solution (composition) with embodiment 1 step 2.3. (2);
8) OPD substrate solution (composition) with embodiment 1 step 2.3. (5);
9) stop buffer (composition) with embodiment 1 step 2.3. (6).
The experimentation of the glycinin double antibodies sandwich ELISA detection by quantitative method of being set up comprises: with the polyclonal antibody bag of Chinese People's Anti-Japanese Military and Political College legumin by the ELISA enzyme plate, order adds: monoclonal antibody, sheep anti-mouse igg/HRP ELIAS secondary antibody, OPD substrate solution, stop buffer that sample to be checked, the legumin cell strain of monoclonal antibody 4B2 CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College are produced, detect the light absorption value (OD of solution in each hole then with microplate reader
492).Can be standard substance with the glycinin, the doubling dilution application of sample be set up typical curve according to the light absorption value reading.Sample well light absorption value comparison typical curve draws accurate glycinin content.Concrete method and operate as follows:
(1) processing of soybean sample:
If sample to be detected is soybean or granulated feed, need be prepared as follows in advance: defibrination behind the soybean peeling, filter back extracted with diethyl ether degreasing, remove residual ether through rotary evaporation, make the degreasing sample, be dissolved in again 0.03mol/L Tris-HCl (beta-mercaptoethanol that contains 0.01mol/L, pH8.0) in.
If sample to be detected is powdery dregs of beans or feed, can directly soybean sample to be measured be dissolved in the 0.03mol/L Tris-HCl (beta-mercaptoethanol that contains 0.01mol/L, pH8.0) in, and magnetic agitation 2h, soybean protein is discharged, so that with the immunological method detection by quantitative actual content of glycinin wherein with the free form.
(2) bag is resisted more: the polyclonal antibody 0.05mol/L NaHCO of the Chinese People's Anti-Japanese Military and Political College legumin behind the purifying
3PH9.6 is made into 2 μ g/ml, and bag is by in 96 hole enzyme plates, and 4 ℃ of (200 μ L/ hole) bags that spend the night are formed insolubilized antibody by solid phases such as polystyrene.
(3) sealing: discard the liquid in the enzyme plate hole.Washing is removed and is not combined with solid phase or in conjunction with behind the not tight antibody, seal with 1% bovine serum albumin confining liquid, and each hole adds confining liquid 150 μ L.Put in the wet box behind 37 ℃ of incubation 1h, with lavation buffer solution washing 3 times, each 90s (be called for short washing, down with) pats dry then.
(4) add testing sample: be cushioned liquid with bag the glycinin standard substance are diluted to eight working concentrations of 0,0.25,0.5,1.0,2.0,4.0,8.0 and 16.0 μ g/mL.Join in the enzyme plate hole by the standard substance of the vertical order of enzyme plate, add each testing sample (each sample is established 10 concentration gradients, and each concentration is established 3 repetitions) then each concentration.Each hole 100 μ L, each concentration gradient or sample are established 3 repetitions at least, establish blank, negative control and positive control simultaneously.Put in the wet box after 37 ℃ of baking ovens are hatched 1h, discard the liquid the hole in, wash, pat dry.
(5) add one anti-(monoclonal antibody): the monoclonal antibody that the legumin cell strain of monoclonal antibody 4B2CGMCC No.2791 of the Chinese People's Anti-Japanese Military and Political College is produced is diluted to best effort concentration (2.5 μ g/mL) with antibody diluent, accurately move to enzyme plate with liquid-transfering gun, each hole 100 μ L puts in the wet box in 37 ℃ of incubation 1h.Discard liquid in the hole, washing pats dry.
(6) add ELIAS secondary antibody: with antibody diluent with ELIAS secondary antibody (sheep anti-mouse igg/HRP) is diluted to working concentration (1: 5000), adds enzyme plate, each hole 100 μ L, place 37 ℃ of incubation 1h after, discard the liquid the hole in, wash, pat dry.
(7) add substrate solution: add fresh preparation OPD substrate to each enzyme mark hole and use liquid 100 μ L, 37 ℃ of incubation 15-30min.
(8) termination reaction: each hole adds stop buffer 50 μ L, stops the substrate color reaction.
(9) result of determination: the light absorption value (OD that under the 492nm wavelength, measures solution in the hole with microplate reader
492), with the pairing light absorption value (OD of the different extent of dilution of standard substance
492) be figure, draw the matched curve equation.Then the light absorption value (OD in testing sample hole
492) bring equation into, can accurately draw the concentration of glycinin.
After testing, the double antibodies sandwich ELISA detection method of this glycinin of researching and developing out:
Sensing range is 0.02 μ g/ml～10 μ g/ml;
Sensitivity is 0.01 μ g/ml;
The variation coefficient is 1.8% for the plate within variance coefficient, and the variation coefficient is 3.3% between plate, and the variation within batch coefficient is 4.0%, and interassay coefficient of variation is 7.2%;
The linearity of the different gradients of sample is evaluated as R
2=0.972.
Claims (10)
1. the monoclonal antibody of glycinin, the legumin cell strain of monoclonal antibody 4B2 of the Chinese People's Anti-Japanese Military and Political College by CGMCC No.2791 is produced by preserving number.
2. preserving number is the legumin cell strain of monoclonal antibody 4B2 of the Chinese People's Anti-Japanese Military and Political College of CGMCC No.2791.
3. the test kit that contains the monoclonal antibody of the described glycinin of claim 1.
4. test kit according to claim 3 is characterized in that: described test kit is an enzyme-linked immunologic detecting kit.
5. with the monoclonal antibody of the described glycinin of claim 1 and the solid phase carrier immune affinity sorbent that obtains of coupling mutually.
6. be the immune affinity chromatographic column of filler with the described immune affinity sorbent of claim 5.
7. the test kit that contains described immune affinity sorbent of claim 5 or the described immune affinity chromatographic column of claim 6.
8. the test kit of the monoclonal antibody of the described glycinin of claim 1 or claim 3 or 4 is following 1) or 2) application:
1) application in detecting glycinin;
2) application in the separation and purification glycinin.
9. the described immune affinity sorbent of claim 5, the described immune affinity chromatographic column of claim 6 or the application of the described test kit of claim 7 in the separation and purification glycinin.
10. the antagonist of glycinin, its activeconstituents is the monoclonal antibody of the described glycinin of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102410058A CN101445558B (en) | 2008-12-24 | 2008-12-24 | Monoclonal antibody of glycinin and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102410058A CN101445558B (en) | 2008-12-24 | 2008-12-24 | Monoclonal antibody of glycinin and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101445558A CN101445558A (en) | 2009-06-03 |
| CN101445558B true CN101445558B (en) | 2011-07-20 |
Family
ID=40741470
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102410058A Expired - Fee Related CN101445558B (en) | 2008-12-24 | 2008-12-24 | Monoclonal antibody of glycinin and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101445558B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102879585A (en) * | 2012-09-28 | 2013-01-16 | 安徽农业大学 | Indirect ELISA (enzyme linked immunosorbent assay) method for detecting soybean antigenic protein serum antibody |
| CN103333862B (en) * | 2013-06-08 | 2014-12-03 | 北京龙科方舟生物工程技术有限公司 | Anti-beta-conglycinin monoclonal antibody and application thereof |
| CN103937750B (en) * | 2014-03-26 | 2015-04-08 | 北京龙科方舟生物工程技术有限公司 | Anti-glycinin monoclonal antibody and application |
| CN103911353B (en) * | 2014-04-18 | 2015-04-08 | 北京龙科方舟生物工程技术有限公司 | Trypsin inhibitor monoclonal antibody and application thereof |
| CN104865388A (en) * | 2015-05-25 | 2015-08-26 | 吉林农业大学 | Establishment method and application of direct ELISA (enzyme-linked immuno sorbent assay) method of glycinin |
| CN109575131A (en) * | 2018-11-28 | 2019-04-05 | 必欧瀚生物技术（合肥）有限公司 | A kind of preparation method of the rabbit polyclonal antibody of pregnancy glycoprotin 3 |
-
2008
- 2008-12-24 CN CN2008102410058A patent/CN101445558B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Jane M Carter et al.Characterisation of polyclonal and monoclonal antibodies against glycinin (11S storage protein) from soya (Glycine max).《Journal of the Science of Food and Agriculture》.1992,第58卷(第1期),第75-82页. * |
| Kyoden Yasumoto et al.Quantitation of soya protein by enzyme linked immunosorbent assay of its characteristic peptide.《Journal of the Science of Food and Agriculture》.1990,第50卷(第3期),第377-389页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101445558A (en) | 2009-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101445558B (en) | Monoclonal antibody of glycinin and application thereof | |
| CN101921731B (en) | Monoclonal antibody of fluoroquinolone medicines as well as preparation method and application thereof | |
| CN103173420B (en) | Hybridoma cell capable of secreting anti-cardiac troponin I monoclonal antibodies and applications thereof | |
| CN105112398A (en) | Preparation of hybridoma cell, monoclonal antibody secreted by hybridoma cell and application of monoclonal antibody | |
| CN101441222B (en) | Method for detecting Beta accompany glycinin and specific antibody and reagent kit thereof | |
| CN105838681B (en) | One plant of anti-dexamethasone monoclonal antibody specific hybridoma cell strain C3 and its application | |
| CN104004717A (en) | Anti-aflatoxin general type monoclonal antibody hybridoma cell line and application thereof | |
| CN101776689A (en) | Immune colloidal gold strip for detecting residue of roxarsone and preparation method thereof | |
| CN104178458B (en) | One plant of Listeria monocytogenes monoclonal antibody hybridoma cell strain and its application | |
| CN108998424A (en) | One plant of aristolochic acid A monoclonal antibody hybridoma cell strain and its application | |
| CN102690788A (en) | Zearalenone anti-idiotypic antibody, preparation method thereof, and application thereof | |
| CN102417895B (en) | Monoclonal antibody for beta-conglycinin beta-subunit and application of monoclonal antibody | |
| CN105754954A (en) | Imidacloprid monoclonal antibody hybridoma cell strain YH5 and application thereof | |
| CN104004718B (en) | One strain anti-Pirlimycin general purpose single monoclonal hybridomas cell line and application thereof | |
| CN106929479B (en) | Vitamin B2 monoclonal antibody hybridoma cell strain GZ-4 and application thereof | |
| CN113046325B (en) | Vitamin K 3 Monoclonal antibody hybridoma cell strain and application thereof | |
| CN105907725B (en) | One plant of anti-hydrocortisone monoclonal antibody specific hybridoma cell strain YH7 and its application | |
| CN103193873A (en) | Immunogen for preparing antibodies used for identification of denatured Bt proteins, antibody prepared by same and application thereof | |
| CN107058240A (en) | One strain of hybridoma strain AB1 and its 2,4,5 trichlorophenoxyacetic acid monoclonal antibodies of generation | |
| CN104311438B (en) | Ractopamine semiantigen, artificial antigen and monoclonal antibody, and preparation methods and applications thereof | |
| CN106636006A (en) | Papaverine monoclonal antibody hybridoma cell strain YH3 and application thereof | |
| CN102841203A (en) | Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN) | |
| CN107043752A (en) | Hybridoma cell strain and the anti-CRH antibody N D19 based on hybridoma cell strain and its application of context of detection | |
| CN101498728B (en) | Reagent kit for detecting o-allyl phenol and its special antibody | |
| CN101633920A (en) | Preparation method of monoclonal antibody of resisting treeshrew IgG |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110720 Termination date: 20171224 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |