CN105181954A - Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof - Google Patents
Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof Download PDFInfo
- Publication number
- CN105181954A CN105181954A CN201510658433.0A CN201510658433A CN105181954A CN 105181954 A CN105181954 A CN 105181954A CN 201510658433 A CN201510658433 A CN 201510658433A CN 105181954 A CN105181954 A CN 105181954A
- Authority
- CN
- China
- Prior art keywords
- antibody
- ovotransferrins
- rabbit
- pillar
- add
- 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.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
Abstract
The invention provides a preparation method of rabbit anti ovotrasferrin allergen polyclone antibody and an immunoassay method thereof. The preparation method comprises the steps that ovotransferrin rabbits are directly immune to New Zealand white rabbits, after immunization is conducted for five times, whole blood is taken from femoral arteries, and ovotransferrin antibody serum is obtained; the antibody peridium amount is optimized, the dilution ratio of the antibody, confining liquid and the PH value of a sample buffer solution are detected, and an indirect competitive ELISA method among the ovotransferrin is established. The antibody prepared through the method is high in potency and good in inhibition rate, and the established method is good in sensitivity and accuracy.
Description
Technical field
The invention belongs to high molecular weight protein immunological technique field; In particular to the preparation of ovotransferrins immune animal, specific antibody and the foundation of immune analysis method.
Background technology
Ovotransferrins (Ovotransferrin, OVTorGald3) be one of the main allergen of egg allergen, its content accounts for 12% of egg white total protein, it is a kind of glycoprotein having ferric ion to combine, ovotransferrins usually has antibacterial activity under form freely, ovotransferrins is made up of two domains, be respectively N territory and C territory, it is a single peptide chain, be made up of 686 amino acid, it can combine with ferric ion, a kind of glycoprotein, containing 15 disulfide bond in each protein molecular, there is anti-oxidation characteristics, relatively less to the anaphylaxis research of ovotransferrins at present, ovotransferrins is comparatively responsive to the change of temperature, high temperature can make its anaphylaxis be subject to serious impact.If with ferric ion, the corrupt split such as zinc ion just can improve its thermal stability after forming complex.
Ovotransferrins is a kind of important transferrins, bears the vital role regulating body ferric ion to balance, can maintain transport and the metabolism of body iron, has the biologically actives such as antibacterial, antiviral, anti-oxidant, immunological regulation.The important albumen that biosome needs.
Ovotransferrins transport iron is comprehensively transported in vivo by ferric ion, after ferric ion being delivered to the position of needs, be convenient to ferric ion be separated, the ferric ion needed for the synthesis of haemoglobin is that it provides, and there are some researches show that ovotransferrins can improve hypoferric anemia symptom.
Secondly ovotransferrins also has antibacterial effect, and it carrys out the breeding of anti-bacteria by the ferric ion needed for chelating bacterial growth; There is antibacterial polypeptide in ovotransferrins.For the effect that the Escherichia coli of pathology, staphylococcus aureus, salmonella etc. can be caused can both to hinder its activity, inhibiting effect can also be played to virus, as viruses such as anti-marek's disease virus, herpesvirals, and have than serum transferrin and the better effect of Lactotransferrin.
Also has oxidation resistant effect, the generation of free radical can be suppressed, oxidative damage is in SOD test, ovotransferrins suppresses the purification specific activity ascorbate of O2-, haemocyanin and known antioxidant higher, the ferritin antioxidation activity that the ovum of the non-bind metal ion of ratio after ovotransferrins and metallic ion combine turns is higher, and the ability of scavenging activated oxygen is the most by force the ovotransferrins in conjunction with Cu2+.
Immunoregulation effect, ovotransferrins can promote the breeding of Bifidobacterium, exist with the effect of Bifidobacterium somatomedin, can promote that body is to the absorption of nutriment, become a kind of important factor of muscle cell nutrition, can promote the growth and differ entiation of cell, thus strengthen the immunity of body, ovotransferrins can regulate chicken macrophage and neutrophil cell, mouse macrophage and splenic lymphocyte in vitro.
Summary of the invention
The object of this invention is to provide the preparation method of a kind of rabbit anti-ovotransferrins anaphylactogen polyclonal antibody, and use the immune analysis method of obtained antibody.Technical scheme provided by the invention is:
A preparation method for ovotransferrins rabbit polyclonal antibody, comprises the following steps:
1) antibody preparation
Immune animal selects the blue large ear rabbit in male west, about 3 months monthly age, body weight about 1.5 kilograms, immunogene is ovotransferrins, immunization method adopts subcutaneous and intramuscular injection, carries out four booster immunizations after initial immunity, and booster immunization was respectively at after initial immunity 2 weeks, 4 weeks and 6 weeks and 8 weeks, five immunity adopt femoral artery to get whole blood after one week, specific practice is:
(1) initial immunity: immunogene and Freund's complete adjuvant are with the volume mixture of 1:1, the glass syringe emulsification good through sealing is abundant, until immunogene and adjuvant mix and the state that is creamy white completely, immunizing dose is 1mg/, carries out animal immune;
(2) booster immunization: immunogene and incomplete Freund's adjuvant equal-volume mixing and emulsifying, carries out animal immune, and immunizing dose is 0.5mg/;
(3) when immunity completes, the blood collected first is solidified 2h at 37 DEG C, be then stored in 4 DEG C of refrigerator overnight, make clot contraction, supernatant is separated out, and get supernatant ,-20 DEG C of Refrigerator stores are for subsequent use;
2) antibody purification
(1) by ultrasonic 20 minutes of all reagent required in experiment;
(2) purification column is balanced: first use flushing line 1-2h, the reagent selected is the phosphate buffer of pH7.4, flow velocity 0.5-1.5mL/min, purification column is got after flushing of pipeline, and a part of ethanolic solution of Feng Zhuyong is slowly poured out, leave the ethanolic solution of post height about 1/3rd; Purification column and protein purification instrument are connected, rinse balance pillar with the phosphate buffer of pH7.4, flow rate set is 0.5-1.5mL/min; When procedures of observation screen medium ultraviolet and conductance two baselines are parallel lines, can stop rushing post;
(3) loading: get the anti-ovotransferrins polyclonal antibody of 0.5-1.5mL rabbit to be purified and phosphate buffer equal-volume dilutes, flow velocity is set to 0.2-0.8mL/min, then by mixed liquor upper prop;
(4) wash-out: after there is foreign protein peak, continuation phosphate buffer rinses pillar, stops after baseline reaches balance; With the glycine-HCI wash buffer pillar of pH2.7, flow velocity 0.5-1.5mL/min, the immunoglobulin (Ig) be now combined with ProteinA-Sepharose4B is eluted;
(5) collect: after adding eluent, just start to collect eluent when baseline starts to change, each peace deferent collection tube collects 0.5-1.5mL liquid, until curve does not change, stops collecting; Collect liquid through spectrophotometer 280nm place reading, discard the collection liquid of absorbance <0.4, regulate antibody rapidly with Tris-HCl immediately by after the liquid blending of absorbance >0.4, make its pH to 7.0;
(6) purification column is processed: after antibody purification terminates, pillar 1-3min is rinsed rapidly with 0.05-0.2mol/L acetic acid, flow velocity 3-8mL/min, and then balance pillar with phosphate buffer, flow velocity 1-8mL/min, measure the pH value flowing out damping fluid with pH test paper, until its display neutrality simultaneously; Be finally that 20% ethanolic solution rinses purification column 20min by mass concentration, and make in pillar, to be full of 20% ethanolic solution, be sealed in 4 DEG C of refrigerators;
(7) antibody is preserved: dialysed three days with PBS under 4 DEG C of environment by the ovotransferrins antibody after purifying, dislysate is the PBS solution of 0.01M, dislysate changes three to four times every day, mix with isopyknic glycerine after antibody after dialysis takes out, save backup in-20 DEG C of refrigerators after packing.
The immune analysis method of the anti-ovotransferrins polyclonal antibody of rabbit of preparation, comprises the steps,
1) bag quilt: with coating buffer dilution ovotransferrins standard solution, be coated in the ELISA Plate of 96 hole polystyrenes, 100 μ L/ holes, discard liquid in hole in 4 DEG C of refrigerators after hatching 12-16h, PBST washing lotion washes plate 3 times, and every 2 minutes once;
2) close: every hole adds 200 μ l, 0.5% skimmed milk powder as confining liquid, in 37 DEG C of baking ovens, close 1h, and take out and discard confining liquid, PBST washing lotion washes plate 3 times, and every 2 minutes once;
3) application of sample: every hole adds 50 μ L ovotransferrins standard solution, add the anti-ovotransferrins polyclonal antibody of 50 μ L rabbit again, the extension rate of antibody is 1:10000, competitive reaction 1h in 37 DEG C of baking ovens, taking-up PBST washing lotion washes plate 4 times, and every 2 minutes once;
4) add ELIAS secondary antibody: the anti-PBS damping fluid of goat-anti rabbit two of horseradish peroxidase-labeled dilutes 10000 times, and 100 μ L/ holes add in ELISA Plate, 37 DEG C of baking oven reaction 30min, PBST washing lotions wash plate 4 times, once per minute;
5) develop the color: 15min need be shifted to an earlier date and substrate A is put in substrate groove, altogether wash plate 5 times, after the 4th time, add substrate B, mixing; Add 100 μ L in every hole, develop the color about 15-20min in 37 DEG C of baking ovens;
6) stop: in every hole, add 50 μ L stop buffer cessation reactions;
7) reading: read absorbance by microplate reader, adopts 450-650nm dual wavelength mode;
8) drawing standard curve.
The solution formula used is as follows:
Coating buffer: 0.05mol/L sodium carbonate-bicarbonate damping fluid, pH is 9.6: accurately take Na respectively
2cO
31.60g, NaHCO
32.91g, then adds distilled water and is settled to 1000mL, finally adjusts pH to 9.6.
Substrate system solution (TMB-hydrogen peroxide urea solution)
Substrate solution A: accurately take anhydrous sodium acetate 8.20g, powder-beta-dextrin 2.50g, be dissolved in the distilled water of 1000mL, then citric acid 3.23g is accurately claimed to regulate pH to 5.0, after all medicines dissolve completely, accurately take carbamide peroxide 428.6mg, and added in the solution prepared.Preserve in 4 DEG C of refrigerators, 15min need be shifted to an earlier date during use and take out, to be restoredly can to use to room temperature.
Substrate solution B: accurately take 100mgTMB and be dissolved in 10mLDMSO, preserves at normal temperature, and deposits in brown bottle dark place.
Substrate solution A and substrate solution B needs to mix before use: get 14.60mL substrate A solution and 0.45mL substrate B solution respectively.
The H of stop buffer: 1.25mol/L
2sO
4solution.
Advantage of the present invention is: antibody titer prepared by the present invention is higher, and inhibiting rate is better, the method sensitivity of foundation and precision better.
Accompanying drawing explanation
The accompanying drawing forming a part of the present invention is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
The typical curve of Fig. 1 for drawing in the immune analysis method described in the embodiment of the present invention one.
Embodiment
It should be noted that, when not conflicting, the embodiment in the present invention and the feature in embodiment can combine mutually.
The present invention is described in detail below in conjunction with embodiment.
Embodiment 1
Immune animal selects the blue large ear rabbit in male west, about 3 months monthly age, body weight about 1.5 kilograms, and immunogene is ovotransferrins,
Immunization method adopts subcutaneous and intramuscular injection, carries out four booster immunizations after initial immunity, and booster immunization was respectively at after initial immunity 2 weeks, 4 weeks and 6 weeks and 8 weeks, and whole blood is got in five immunity one week afterwards employing femoral artery, and specific practice is:
(1) initial immunity: immunogene and Freund's complete adjuvant are with the volume mixture of 1:1, the glass syringe emulsification good through sealing is abundant, until immunogene and adjuvant mix and the state that is creamy white completely, immunizing dose is 1mg/, carries out animal immune;
(2) booster immunization: immunogene and incomplete Freund's adjuvant equal-volume mixing and emulsifying, carries out animal immune; Immunity amount is 0.5mg/;
(3) when immunity completes, the blood collected first is solidified 2h at 37 DEG C, be then stored in 4 DEG C of refrigerator overnight, make clot contraction, supernatant is separated out, and get supernatant ,-20 DEG C of Refrigerator stores are for subsequent use.
Antibody purification
(1) by ultrasonic 20 minutes of all reagent required in experiment;
(2) purification column is balanced: first use flushing line 1-2h, the reagent selected is the phosphate buffer of pH7.4, flow velocity 0.5-1.5mL/min, purification column is got after flushing of pipeline, and a part of ethanolic solution of Feng Zhuyong is slowly poured out, leave the ethanolic solution of post height about 1/3rd; Purification column and protein purification instrument are connected, rinse balance pillar with the phosphate buffer of pH7.4, flow rate set is 0.5-1.5mL/min; When procedures of observation screen medium ultraviolet and conductance two baselines are parallel lines, can stop rushing post;
(3) loading: get the anti-ovotransferrins polyclonal antibody of 0.5-1.5mL rabbit to be purified and phosphate buffer equal-volume dilutes, flow velocity is set to 0.2-0.8mL/min, then by mixed liquor upper prop;
(4) wash-out: after there is foreign protein peak, continuation phosphate buffer rinses pillar, stops after baseline reaches balance; With the glycine-HCI wash buffer pillar of pH2.7, flow velocity 0.5-1.5mL/min, the immunoglobulin (Ig) be now combined with ProteinA-Sepharose4B is eluted;
(5) collect: after adding eluent, just start to collect eluent when baseline starts to change, each peace deferent collection tube collects 0.5-1.5mL liquid, until curve does not change, stops collecting; Collect liquid through spectrophotometer 280nm place reading, discard the collection liquid of absorbance <0.4, regulate antibody rapidly with Tris-HCl immediately by after the liquid blending of absorbance >0.4, make its pH to 7.0;
(6) purification column is processed: after antibody purification terminates, pillar 1-3min is rinsed rapidly with 0.05-0.2mol/L acetic acid, flow velocity 3-8mL/min, and then balance pillar with phosphate buffer, flow velocity 1-8mL/min, measure the pH value flowing out damping fluid with pH test paper, until its display neutrality simultaneously; Be finally that 20% ethanolic solution rinses purification column 20min by mass concentration, and make in pillar, to be full of 20% ethanolic solution, be sealed in 4 DEG C of refrigerators;
(7) antibody is preserved: dialysed three days with PBS under 4 DEG C of environment by the ovotransferrins antibody after purifying, dislysate is the PBS solution of 0.01M, dislysate changes three to four times every day, mix with isopyknic glycerine after antibody after dialysis takes out, save backup in-20 DEG C of refrigerators after packing.
The ovotransferrins rabbit polyclonal antibody of above-mentioned preparation is used for the foundation of indirect competition method.
Ovotransferrins indirect competitive ELISA method condition determination preferred:
The optimization of antigen coated amount and antibody dilution multiple:
Ovotransferrins package amount is selected to be 0.1 μ g/ hole, 0.05 μ g/ hole and 0.03 μ g/ hole, the dilution ratio that rabbit resists is 1:10000,1:15000,1:20000, application chessboard method carries out indirect competitive ELISA, measure maximum absorbance value, calculate corresponding inhibiting rate, draw inhibiting rate curve, calculate IC50, its result is as shown in table 1.Experimentally requirement, chooses maximum absorbance value at about 0.8-1.2, and what the IC50 of its correspondence was minimum is combined as best package amount and antibody dilution multiple.Therefore, be 0.03 μ g/ hole according to the known best package amount of data result in table, antibody dilution ratio is 1:10000.
The impact that table 1 package amount and antibody dilution multiple detect ovotransferrins indirect competitive enzyme-linked immunosorbent
Table1EffectofquantityofcaptureantibodyanddetectantibodyonindirectcompetitionELISA
The optimization of confining liquid:
The experimental selection skimmed milk powder of 0.5%, the skimmed milk powder of 1%, skimmed milk powder, the 1%BSA solution of 5%, 0.5% fish glue from skin, 1% gelatin, 0.5% casein is respectively as confining liquid.As shown in Table 3-5.As shown in table 2, when adopting the milk powder of 0.5% to close, blank value is minimum, and Control value is minimum at about 1.2, IC50, therefore have selected the milk powder of sealing effect good 0.5% as confining liquid.
Mean absorbance values blank under the different confining liquid of table 2
Table2Absorptionvaluesatthedifferentblockingbuffer
(3) optimization of sample buffer pH
Have table 3 known, when phosphate buffer pH is 7.4, and Control value is minimum in about 1.2, IC50 value, and therefore the phosphate buffer of experimental selection pH7.4 is sample diluting liquid.
The impact that table 3pH value detects indirect competitive enzyme-linked immunosorbent
Table3EffectofpHonindirectcompetitionELISA
According to the result that above-mentioned optimization obtains, with the logarithm of ovotransferrins concentration for horizontal ordinate, with ovotransferrins concentration value for X-axis, corresponding inhibiting rate is Y-axis, obtains ovotransferrins indirect competitive ELISA typical curve, see Fig. 1 through semilog mapping.
The antibody of above-mentioned preparation can be used for the immune detection of ovotransferrins, and its method is as follows:
(1) bag quilt: with coating buffer dilution ovotransferrins standard solution, be coated in 96 hole polystyrene ELISA Plate, 100 μ L/ holes, discard liquid in hole in 4 DEG C of refrigerators after hatching 12-16h, PBST washing lotion washes plate 3 times, and every 2 minutes once.
(2) close: every hole adds 200 μ l, 0.5% skimmed milk powder as confining liquid, in 37 DEG C of baking ovens, close 1h, and take out and discard confining liquid, PBST washing lotion washes plate 3 times, and every 2 minutes once.
(3) application of sample: every hole adds 50 μ L ovotransferrins standard solution, then adds the anti-ovotransferrins antibody of 50 μ L rabbit, and competitive reaction 1h in 37 DEG C of baking ovens, taking-up PBST washing lotion washes plate 4 times, and every 2 minutes once.
(4) add ELIAS secondary antibody: the anti-PBS of goat-anti rabbit two of HRP mark dilutes 10000 times, and 100 μ L/ holes add in ELISA Plate, 37 DEG C of baking oven reaction 30min, PBST washing lotions wash plate 4 times, once per minute.
(5) develop the color: 15min need be shifted to an earlier date and substrate A is put in substrate groove, altogether wash plate 5 times, after the 4th time, add substrate B, mixing.Add 100 μ L in every hole, develop the color about 15-20min in 37 DEG C of baking ovens.
(6) stop: in every hole, add 50 μ L stop buffer cessation reactions.
(7) reading: read absorbance (OD value) by microplate reader, adopts dual wavelength mode (450-650nm).
(8) drawing standard curve: the X-axis of inhibiting rate curve map is ovotransferrins concentration value, and Y-axis is corresponding inhibiting rate, typical curve is typical S type curve, and IC50 is defined as ovotransferrins concentration corresponding when inhibiting rate is 50%.
Sensitivity: under the optimal conditions determined, establishes the typical curve of ovotransferrins indirect competitive ELISA method as shown in Figure 1, can draw the sensitivity of the method from figure, and its 503nhibiting concentration IC50 value is 619.26 ± 65.6ng/mL.
Specificity: this experiment uses the ovotransferrins antibody albumen to 10 kinds of daily easy sensitization food to carry out cross reaction detection, and object is the specificity checking antibody, and cross reacting rate is less, shows that the specificity of antibody is better.As shown in table 4, ovotransferrins has with albumin and intersects, and it and other several frequently seen allergen proteins do not have cross reaction.
Table 4 ovotransferrins antibody and other cross reactions
Table4Cross-reactivityofantibodyofOVTwithselectedcommonfoods
Precision:
Precision refers to that, under the test condition of regulation, same homogeneous sample, through the degree of closeness repeatedly between sampling and measuring acquired results.In ELISA detects, the precision coefficient of variation is weighed.The coefficient of variation represents with (CV%), represents the degree of stochastic error size in measurement result.
The present invention selects the ovotransferrins solution of 6 variable concentrations to analyze, and examines or check in plate making a variation between variation and plate respectively, carries out precision test to the ovotransferrins indirect competitive enzyme-linked immunosorbent detection method set up.
Variation in plate: select the ovotransferrins of 6 variable concentrations to carry out the analysis of indirect competitive enzyme-linked immunosorbent detection method, each concentration does 5 repetitions in same ELISA Plate, according to the enzyme-linked immune detection method set up, each concentration is analyzed, measure its absorbance, and calculate inhibiting rate, the coefficient of variation.Result is as following table 5.
Variation (n=5) in table 5 ovotransferrins indirect competitive ELISA plate
Table5Variancein-plateoficELISAforOVT(n＝5)
Make a variation between plate: the ovotransferrins standard solution getting 6 variable concentrations, carry out the analysis of indirect competitive enzyme-linked immunosorbent detection method.Each concentration carries out an indirect competitive ELISA analysis every day, and the inhibiting rate recorded for totally 5 times for comprehensive 5 days also calculates the coefficient of variation, the results are shown in Table 6.
(n=5) is made a variation between table 6 ovotransferrins indirect competitive ELISA plate
Table6Varianceinter-plateoficELISAforOVT(n＝5)
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; within all spirit in the invention and principle, any amendment done, equivalent replacement, improvement etc., within the protection domain that all should be included in the invention.
Claims (3)
1. a preparation method for the anti-ovotransferrins polyclonal antibody of rabbit, is characterized in that: comprise the following steps:
1) antibody preparation
Immune animal selects the blue large ear rabbit in male west, about 3 months monthly age, body weight about 1.5 kilograms, immunogene is ovotransferrins, immunization method adopts subcutaneous and intramuscular injection, carries out four booster immunizations after initial immunity, and booster immunization was respectively at after initial immunity 2 weeks, 4 weeks and 6 weeks and 8 weeks, five immunity adopt femoral artery to get whole blood after one week, specific practice is:
(1) initial immunity: immunogene and Freund's complete adjuvant with 1: 1 volume mixture, the glass syringe emulsification good through sealing is abundant, until immunogene and adjuvant mix and the state that is creamy white completely, immunizing dose is 1mg/, carries out animal immune;
(2) booster immunization: immunogene and incomplete Freund's adjuvant equal-volume mixing and emulsifying, carries out animal immune, and immunizing dose is 0.5mg/;
(3) when immunity completes, the blood collected first is solidified 2h at 37 DEG C, be then stored in 4 DEG C of refrigerator overnight, make clot contraction, supernatant is separated out, and get supernatant ,-20 DEG C of Refrigerator stores are for subsequent use;
2) antibody purification
(1) by ultrasonic 20 minutes of all reagent required in experiment;
(2) purification column is balanced: first use flushing line 1-2h, the reagent selected is the phosphate buffer of pH7.4, flow velocity 0.5-1.5mL/min, purification column is got after flushing of pipeline, and a part of ethanolic solution of Feng Zhuyong is slowly poured out, leave the ethanolic solution of post height about 1/3rd; Purification column and protein purification instrument are connected, rinse balance pillar with the phosphate buffer of pH7.4, flow rate set is 0.5-1.5mL/min; When procedures of observation screen medium ultraviolet and conductance two baselines are parallel lines, can stop rushing post;
(3) loading: get the anti-ovotransferrins polyclonal antibody of 0.5-1.5mL rabbit to be purified and phosphate buffer equal-volume dilutes, flow velocity is set to 0.2-0.8mL/min, then by mixed liquor upper prop;
(4) wash-out: after there is foreign protein peak, continuation phosphate buffer rinses pillar, stops after baseline reaches balance; With the glycine-HCI wash buffer pillar of pH2.7, flow velocity 0.5-1.5mL/min, the immunoglobulin (Ig) be now combined with ProteinA-Sepharose4B is eluted;
(5) collect: after adding eluent, just start to collect eluent when baseline starts to change, each peace deferent collection tube collects 0.5-1.5mL liquid, until curve does not change, stops collecting; Collect liquid through spectrophotometer 280nm place reading, discard the collection liquid of absorbance < 0.4, regulate antibody rapidly with Tris-HCl immediately by after the liquid blending of absorbance > 0.4, make its pH to 7.0;
(6) purification column is processed: after antibody purification terminates, pillar 1-3min is rinsed rapidly with 0.05-0.2mol/L acetic acid, flow velocity 3-8mL/min, and then balance pillar with phosphate buffer, flow velocity 1-8mL/min, measure the pH value flowing out damping fluid with pH test paper, until its display neutrality simultaneously; Be finally that 20% ethanolic solution rinses purification column 20min by mass concentration, and make in pillar, to be full of 20% ethanolic solution, be sealed in 4 DEG C of refrigerators;
(7) antibody is preserved: dialysed three days with PBS under 4 DEG C of environment by the ovotransferrins antibody after purifying, dislysate is the PBS solution of 0.01M, dislysate changes three to four times every day, mix with isopyknic glycerine after antibody after dialysis takes out, save backup in-20 DEG C of refrigerators after packing.
2. an immune analysis method for the rabbit anti-ovotransferrins anaphylactogen polyclonal antibody using the preparation method as described in claims 1 to 3 to prepare, is characterized in that: comprise the steps,
1) bag quilt: with coating buffer dilution ovotransferrins standard solution, be coated in the ELISA Plate of 96 hole polystyrenes, 100 μ L/ holes, discard liquid in hole in 4 DEG C of refrigerators after hatching 12-16h, PBST washing lotion washes plate 3 times, and every 2 minutes once;
2) close: every hole adds 200 μ l, 0.5% skimmed milk powder as confining liquid, in 37 DEG C of baking ovens, close 1h, and take out and discard confining liquid, PBST washing lotion washes plate 3 times, and every 2 minutes once;
3) application of sample: every hole adds 50 μ L ovotransferrins standard solution, add the anti-ovotransferrins polyclonal antibody of 50 μ L rabbit again, the extension rate of antibody is 1:10000, competitive reaction 1h in 37 DEG C of baking ovens, taking-up PBST washing lotion washes plate 4 times, and every 2 minutes once;
4) add ELIAS secondary antibody: the anti-PBS damping fluid of goat-anti rabbit two of horseradish peroxidase-labeled dilutes 10000 times, and 100 μ L/ holes add in ELISA Plate, 37 DEG C of baking oven reaction 30min, PBST washing lotions wash plate 4 times, once per minute;
5) develop the color: 15min need be shifted to an earlier date and substrate A is put in substrate groove, altogether wash plate 5 times, after the 4th time, add substrate B, mixing; Add 100 μ L in every hole, develop the color about 15-20min in 37 DEG C of baking ovens;
6) stop: in every hole, add 50 μ L stop buffer cessation reactions;
7) reading: read absorbance by microplate reader, adopts 450-650nm dual wavelength mode;
8) drawing standard curve.
3. immune analysis method according to claim 2, is characterized in that: step 1) in, the package amount of ovotransferrins is 0.03 μ g/ hole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510658433.0A CN105181954A (en) | 2015-10-12 | 2015-10-12 | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510658433.0A CN105181954A (en) | 2015-10-12 | 2015-10-12 | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105181954A true CN105181954A (en) | 2015-12-23 |
Family
ID=54904176
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510658433.0A Pending CN105181954A (en) | 2015-10-12 | 2015-10-12 | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105181954A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112326567A (en) * | 2020-10-09 | 2021-02-05 | 天津科技大学 | Optimized extraction method of pumpkin antioxidant active ingredients and verification method of antioxidant activity |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003261452A (en) * | 2002-03-11 | 2003-09-16 | National Agricultural Research Organization | Immune response inhibitor |
| WO2004015123A1 (en) * | 2002-08-09 | 2004-02-19 | Origen Therapeutics | Tissue specific expression of exogenous proteins in transgenic chickens |
| US20090165155A1 (en) * | 2005-02-09 | 2009-06-25 | Lei Zhu | Tissue Specific Expression of Exogenous Proteins in Transgenic Chickens |
| CN102650640A (en) * | 2012-04-24 | 2012-08-29 | 中国人民解放军第四军医大学 | Method for detecting allergen ovomucoid or ovotransferrin in influenza vaccine quantificationally |
| CN102993299A (en) * | 2001-09-05 | 2013-03-27 | 日本肉类批发商株式会社 | Food allergens, method of detecting food allergens and method of detecting food allergy-inducing foods |
| CN104945507A (en) * | 2015-05-22 | 2015-09-30 | 天津科技大学 | Preparation method of ovomucoid rabbit polyclonal antibody and immunoassay method |
-
2015
- 2015-10-12 CN CN201510658433.0A patent/CN105181954A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102993299A (en) * | 2001-09-05 | 2013-03-27 | 日本肉类批发商株式会社 | Food allergens, method of detecting food allergens and method of detecting food allergy-inducing foods |
| JP2003261452A (en) * | 2002-03-11 | 2003-09-16 | National Agricultural Research Organization | Immune response inhibitor |
| WO2004015123A1 (en) * | 2002-08-09 | 2004-02-19 | Origen Therapeutics | Tissue specific expression of exogenous proteins in transgenic chickens |
| US20090165155A1 (en) * | 2005-02-09 | 2009-06-25 | Lei Zhu | Tissue Specific Expression of Exogenous Proteins in Transgenic Chickens |
| CN102650640A (en) * | 2012-04-24 | 2012-08-29 | 中国人民解放军第四军医大学 | Method for detecting allergen ovomucoid or ovotransferrin in influenza vaccine quantificationally |
| CN104945507A (en) * | 2015-05-22 | 2015-09-30 | 天津科技大学 | Preparation method of ovomucoid rabbit polyclonal antibody and immunoassay method |
Non-Patent Citations (3)
| Title |
|---|
| CHURCH WR等: "Monoclonal antibodies to the amino- and carboxyl-terminal domains of ovotansferrin", 《HYBRIDOMA》 * |
| GAUTRON J: "Ovotransferrin is a matrix protein of the hen eggshenll membranes and basal calcified layer", 《CONNECT TISSUE RES.》 * |
| 佟平等: "高效特异的鸡蛋过敏原卵转铁蛋白多克隆抗体的制备", 《食品科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112326567A (en) * | 2020-10-09 | 2021-02-05 | 天津科技大学 | Optimized extraction method of pumpkin antioxidant active ingredients and verification method of antioxidant activity |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102220286B (en) | Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof | |
| CN101993855B (en) | Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof | |
| JP2010517529A5 (en) | ||
| CN106645681B (en) | A kind of blocking agent kit and its application method for immunochromatographic measurement | |
| CN103575889B (en) | A kind of test strips and method detecting vancomycin | |
| CN101799472A (en) | Diethylstilbestrol detection kit and detection method | |
| CN101592661B (en) | Brucellosis antibody competitive enzyme-linked immunosorbent assay reagent kit | |
| CN106093381A (en) | Rod method toxin rod method phenol monomethyl ether colloidal gold immuno-chromatography test paper strip | |
| CN109917126A (en) | A method of test strips, the preparation method of imidacloprid haptens and the detection Determination of Imidacloprid Residue of detection imidacloprid | |
| CN105116148A (en) | Test strip and method for detecting sodium pentachlorophenate | |
| CN101955536B (en) | Anti-sulfonamides antibody and application thereof | |
| CN100501405C (en) | ELISA kit for detecting penicillin G and detection method thereof | |
| CN103575887B (en) | A kind of test card and application thereof detecting aflatoxin B1 | |
| CN103513035B (en) | A kind of test strips detecting Aflatoxins M1 and method | |
| CN101241135B (en) | ELISA kit for detecting chlopyrifos residue and method of use thereof | |
| CN106831498A (en) | Furacilin metabolite SEM derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN105181954A (en) | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof | |
| CN103105490B (en) | A kind of kit and method detecting tetracycline medication | |
| CN105399639A (en) | Tyramine artificial antigen and antibody, and preparation methods and application thereof | |
| CN107118159A (en) | Cistofuran metabolite AHD derivatizations haptens, the preparation method and applications of artificial antigen | |
| CN103255109A (en) | Hybridoma cell strain AFG1G6, its monoclonal antibody and aflatoxin G1 immunochromatographic test strip | |
| CN103424550B (en) | A kind of kit of chlorine detection mycin and method | |
| CN104945507A (en) | Preparation method of ovomucoid rabbit polyclonal antibody and immunoassay method | |
| CN101962359A (en) | Hapten, artifical antigen and antibody of enrofloxacin and preparation method as well as application thereof | |
| CN101955542B (en) | Immunoassay kit and special antibody for detecting fumonisins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151223 |