CN104945507A - Preparation method of ovomucoid rabbit polyclonal antibody and immunoassay method - Google Patents
Preparation method of ovomucoid rabbit polyclonal antibody and immunoassay method Download PDFInfo
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Abstract
The invention provides a preparation method of an ovomucoid rabbit polyclonal antibody and an immunoassay method. The preparation method comprises the following main steps: directly immunizing a new Zealand white rabbit by using ovomucoid for five times, and then taking whole blood from arteria femoralis, so that an ovomucoid antibody serum is obtained; and through optimizing the coating amount of the antibody, detecting the dilution ratio of the antibody and the pH values of confining liquid and a sample buffer solution, so that an ovomucoid indirect competitive ELISA method is established. The antibody prepared according to the invention is relatively high in potency and relatively good in inhibition rate, and the established method is relatively good in sensitivity and precision.
Description
Technical field
The invention belongs to high molecular weight protein immunological technique field, especially relate to a kind of preparation method and immune analysis method of ovomucoid rabbit polyclonal antibody.
Background technology
Ovomucoid is made up of 186 amino acid, its molecular mass is about 28DK, ovomucoid contains 3 independently homeodomains, and these three functional domains are one in front and one in back continuously arranged, and each functional domain couples together by intramolecular disulfide linkage.Containing 9 intramolecular disulfide bonds in ovomucoid molecule, also have the glycosyl moiety of 20% ~ 25% in addition, what in three regions, sensitization was the strongest is then the 3rd region.The content of ovomucoid in egg white is the highest, and sensitization is also the strongest.
Due to containing glycosyl moiety in ovomucoid, so the stability of ovomucoid is the highest, it is to tryptic degraded quite stable, in addition to thermal treatment also quite stable.Research finds, the susceptibility of short duration to egg, relevant with the lgE content of the decision base of ovomucoid conformation.Ovomucoid causes immunoreactive composition, after taking in ovomucoid in body, can produce more IgG and IgE to resist it in blood.Existing ovomucoid antibody mostly is monoclonal antibody, less to the research of polyclonal antibody, is necessary to research and develop a kind of polyclonal antibody, to improve sensitivity and the specificity of antibody.
Summary of the invention
In view of this, the present invention is intended to the preparation method and the immune analysis method that propose a kind of ovomucoid rabbit polyclonal antibody, to improve sensitivity and the specificity of antibody.
For achieving the above object, technical scheme of the present invention is achieved in that
A preparation method for ovomucoid rabbit polyclonal antibody, comprises immunologic process, antibody purification procedures two steps;
In wherein said immunologic process, immune animal selects rabbit, and immunogen is ovomucoid, and immunization method adopts subcutaneous and intramuscular injection, carries out several times booster immunization after initial immunity, and immunity adopts femoral artery to get whole blood after one week, preserve; Described antibody purification procedures uses purification column and protein purification instrument to realize.
Preferably, in described immunologic process, carry out four booster immunizations after initial immunity, booster immunization was respectively at after initial immunity 2 weeks, 4 weeks and 6 weeks and 8 weeks, and five immunity adopt femoral artery to get whole blood after one week, preserve.
Preferably, described immunologic process specifically comprises the steps,
1) initial immunity: immunogen and Freund's complete adjuvant are with the volume mixture of 1:1, the glass syringe emulsification good through stopping property is abundant, until immunogen and adjuvant mix and the state that is creamy white completely, immunizing dose is 1mg/, carries out animal immune;
2) booster immunization: immunogen and Freund's incomplete 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 blood clot retraction, supernatant liquor is separated out, and get supernatant ,-20 DEG C of Refrigerator stores are for subsequent use.
Preferably, described antibody purification procedures, specifically comprises the steps:
1) by ultrasonic 20 minutes of all reagent required in experiment, bubble is got rid of;
2) purification column is balanced: first use flushing pipeline 1-2h, the reagent selected is the phosphoric acid buffer of pH 7.4, flow velocity 0.5-1.5mL/min; Get purification column after flushing of pipeline, and slowly poured out by a part of ethanolic soln of Feng Zhuyong, leave the ethanolic soln of post height 1/3rd, object prevents pure for post dry in purge process; Purification column and protein purification instrument are connected, rinse balance pillar with the phosphoric acid buffer of pH 7.4, 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, the general hours of this process.
3) loading: get 0.5-1.5mL ovomucoid rabbit polyclonal antibody serum to be purified and phosphoric acid buffer equal-volume dilutes, flow velocity is set to 0.2-0.8mL/min, then by mixed solution upper prop.On the binding site being adsorbed on Protein A-Sepharose 4B of the specific for immunoglobulin in antiserum(antisera), and other impurity such as foreign protein, fat can not be adsorbed, can flow out along with damping fluid, now can demonstrate foreign protein peak in purifying instrument ultraviolet conductance figure;
4) wash-out: after there is foreign protein peak, continuation phosphoric acid buffer rinses pillar, stops after baseline reaches balance; With the glycine-HCI wash buffer pillar of pH 2.7, flow velocity 0.5-1.5mL/min, the immunoglobulin (Ig) be now combined with Protein A-Sepharose 4B is eluted;
5) collect: after adding elutriant, note the change of instrument ultraviolet conductance figure line, just start to collect elutriant 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 buffered saline 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 soln rinses purification column 20min by mass concentration, and make in pillar, to be full of 20% ethanolic soln, be sealed in 4 DEG C of refrigerators;
7) antibody is preserved: dialysed three days under 4 DEG C of environment by the ovomucoid antibody after purifying, dialyzate is the PBS solution of 0.01M, dialyzate changes three to four times every day, mixes, save backup after packing in-20 DEG C of refrigerators after taking out with isopyknic glycerol.
Present invention also offers a kind of immune analysis method of the ovomucoid rabbit polyclonal antibody using preparation method as above to prepare, comprise the steps,
1) bag quilt: with coating buffer dilution ovomucoid standardized solution, be coated in the enzyme 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 ovomucoid standardized solution, then adds 50 μ L ovomucoid rabbit polyclonal antibodies, and competing 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 enzyme plate, 37 DEG C of baking oven reaction 30min, PBST washing lotions wash plate 4 times, and per minute once.
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 termination reactions;
7) reading: read absorbance by microplate reader, adopts 450-650nm dual wavelength mode.
8) drawing standard curve: the X-axis of inhibiting rate graphic representation is ovomucoid concentration value, and Y-axis is corresponding inhibiting rate, typical curve is typical S type curve, and IC50 is defined as ovomucoid concentration corresponding when inhibiting rate is 50%;
Further, the solution formula of above use 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 sodium acetate, anhydrous 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 Urea 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 100mg TMB and be dissolved in 10mL DMSO, 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.
Relative to prior art, the preparation method of ovomucoid rabbit polyclonal antibody of the present invention and immune analysis method, have following advantage:
(1) antibody titer prepared by the preparation method of ovomucoid rabbit polyclonal antibody of the present invention is high, and inhibiting rate is better;
(2) antibody prepared by the preparation method of ovomucoid rabbit polyclonal antibody of the present invention has good sensitivity, and IC50 reaches 649.13 ± 35.1ng/mL;
(3) immune analysis method provided by the invention and indirect competitive ELISA method (enzyme-linked immunosorbent assay) easy and simple to handle, quick, there is higher sensitivity and specificity, its specificity is decided by the specificity of immunological response, and the affinity of antibody is depended in its sensitivity.
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 one
A preparation method for ovomucoid rabbit polyclonal antibody, is characterized in that: comprise immunologic process, antibody purification procedures two steps;
In wherein said immunologic process, immune animal selects rabbit, immunogen is ovomucoid, immunization method adopts subcutaneous and intramuscular injection, four booster immunizations are carried out after initial immunity, booster immunization was respectively at after initial immunity 2 weeks, 4 weeks and 6 weeks and 8 weeks, and five immunity adopt femoral artery to get whole blood after one week, preserve.
Described antibody purification procedures, specifically comprises the steps:
1) by ultrasonic 20 minutes of all reagent required in experiment, bubble is got rid of;
2) purification column is balanced: first use flushing pipeline 1-2h, the reagent selected is the phosphoric acid buffer of pH 7.4, flow velocity 1.0mL/min; Get purification column after flushing of pipeline, and a part of ethanolic soln of Feng Zhuyong is slowly poured out, leave the ethanolic soln of post height 1/3rd; Purification column and protein purification instrument are connected, rinse balance pillar with the phosphoric acid buffer of pH 7.4, flow rate set is 1mL/min; When procedures of observation screen medium ultraviolet and conductance two baselines are parallel lines, can stop rushing post.
3) loading: get 1mL ovomucoid rabbit polyclonal antibody serum to be purified and phosphoric acid buffer equal-volume dilutes, flow velocity is set to 0.5mL/min, then by mixed solution upper prop, can demonstrate foreign protein peak in purifying instrument ultraviolet conductance figure;
4) wash-out: after there is foreign protein peak, continuation phosphoric acid buffer rinses pillar, stops after baseline reaches balance; With the glycine-HCI wash buffer pillar of pH 2.7, flow velocity 1mL/min, the immunoglobulin (Ig) be now combined with Protein A-Sepharose 4B is eluted;
5) collect: after adding elutriant, note the change of instrument ultraviolet conductance figure line, just start to collect elutriant when baseline starts to change, each peace deferent collection tube collects 1mL 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, rinse rapidly pillar 2min with 0.1mol/L acetic acid, flow velocity 5mL/min, then pillar is balanced with phosphate buffered saline buffer, flow velocity 1mL/min, measures the pH value flowing out damping fluid simultaneously with pH test paper, until its display neutrality; Be finally that 20% ethanolic soln rinses purification column 20min by mass concentration, and make in pillar, to be full of 20% ethanolic soln, be sealed in 4 DEG C of refrigerators;
7) antibody is preserved: dialysed three days under 4 DEG C of environment by the ovomucoid antibody after purifying, dialyzate is the PBS solution of 0.01M, dialyzate changes three to four times every day, mixes, save backup after packing in-20 DEG C of refrigerators after taking out with isopyknic glycerol.
An immune analysis method for the ovomucoid rabbit polyclonal antibody using preparation method as above to prepare, comprises the steps,
1) bag quilt: with coating buffer dilution ovomucoid standardized solution, be coated in the enzyme 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 ovomucoid standardized solution, then adds 50 μ L ovomucoid rabbit polyclonal antibodies, and competing 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 enzyme plate, 37 DEG C of baking oven reaction 30min, PBST washing lotions wash plate 4 times, and per minute once.
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 termination reactions;
7) reading: read absorbance by microplate reader, adopts 450-650nm dual wavelength mode.
8) drawing standard curve, with the logarithm of ovomucoid concentration for X-coordinate, with ovomucoid concentration value for X-axis, corresponding inhibiting rate is Y-axis, obtains ovomucoid indirect competitive ELISA typical curve, see accompanying drawing 1 through semilog mapping.
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 sodium acetate, anhydrous 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 Urea 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 100mg TMB and be dissolved in 10mL DMSO, 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.
Sensitivity: under the optimal conditions determined, establishes the typical curve of ovomucoid indirect competitive ELISA method as shown in Figure 1, can draw the sensitivity of the method from figure, its 503nhibiting concentration IC
50value is 649.13 ± 35.1ng/mL.
Specificity: this experiment uses the ovomucoid 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 1, ovomucoid and other several frequently seen allergen proteins do not have cross reaction.
Table 1 ovomucoid antibody and other cross reactions
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 variation coefficient is weighed.The variation coefficient represents with (CV%), represents the degree of random error size in measuring result.
The present invention selects the ovomucoid solution of 7 different concns to analyze, and examines or check in plate making a variation between variation and plate respectively, carries out precision test to the ovomucoid indirect competitive enzyme-linked immunosorbent detection method set up.
Variation in plate: select the ovomucoid of 7 different concns to carry out the analysis of indirect competitive enzyme-linked immunosorbent detection method, each concentration does 5 repetitions in same enzyme plate, according to the enzyme-linked immune detection method set up, each concentration is analyzed, measure its absorbance, and calculate inhibiting rate, the variation coefficient.Result is as following table 2.
Variation in table 2 ovomucoid indirect competitive ELISA plate
Make a variation between plate: the ovomucoid standardized solution getting 7 different concns, 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 variation coefficient, the results are shown in Table 3.
(n=5) is made a variation between table 3 ovomucoid indirect competitive ELISA plate
The simultaneous test of ovomucoid indirect competitive ELISA method condition determination:
(1) contrast of antigen coated amount and antibody dilution multiple:
Ovomucoid 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 1:25000,1:30000,1:35000,1:40000, application chessboard method carries out indirect competitive ELISA, measure maximum absorbance value, calculate corresponding inhibiting rate, draw inhibiting rate curve, calculate IC
50, its result is as shown in table 1.Experimentally requirement, chooses maximum absorbance value at about 0.8-1.2, the IC of its correspondence
50minimum is combined as best package amount and antibody dilution multiple.Therefore, be 0.05 μ g/ hole according to the known best package amount of data results in table, antibody dilution is 1:40000.
The impact that the antigen coated amount of table 1 and antibody dilution multiple detect Saliva Orthana indirect competitive enzyme-linked immunosorbent
(note: A
controlabsorbance corresponding when only adding 50 μ L antibody and 50 μ LPBS solution in finger-hole)
(2) contrast 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 2, when adopting the milk powder of 0.5% to close, blank value is minimum, and A
controlminimum at about 1.2, IC50, therefore the milk powder of 0.5% is as confining liquid, and its sealing effect is good.
The contrast of table 2 confining liquid
(note: A
controlabsorbance corresponding when only adding 50 μ L antibody and 50 μ LPBS solution in finger-hole)
(3) contrast of sample buffer pH
As shown in Table 3, when phosphoric acid buffer pH is 7.4, and A
controlminimum in about 1.2, IC50 value, therefore the phosphate buffered saline buffer of pH7.4 is sample diluting liquid, has good experiment effect.
(note: A
controlabsorbance corresponding when only adding 50 μ L antibody and 50 μ LPBS solution in finger-hole)
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 (6)
1. a preparation method for ovomucoid rabbit polyclonal antibody, is characterized in that: comprise immunologic process, antibody purification procedures two steps;
In wherein said immunologic process, immune animal selects rabbit, and immunogen is ovomucoid, and immunization method adopts subcutaneous and intramuscular injection, carries out several times booster immunization after initial immunity, and immunity adopts femoral artery to get whole blood after one week, preserve; Described antibody purification procedures uses purification column and protein purification instrument to realize.
2. the preparation method of ovomucoid rabbit polyclonal antibody according to claim 1, it is characterized in that: in described immunologic process, four booster immunizations are carried out after initial immunity, 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, preserve.
3. the preparation method of ovomucoid rabbit polyclonal antibody according to claim 2, is characterized in that: described immunologic process specifically comprises the steps,
1) initial immunity: immunogen and Freund's complete adjuvant are with the volume mixture of 1:1, the glass syringe emulsification good through stopping property is abundant, until immunogen and adjuvant mix and the state that is creamy white completely, immunizing dose is 1mg/, carries out animal immune;
2) booster immunization: immunogen and Freund's incomplete 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 blood clot retraction, supernatant liquor is separated out, and get supernatant ,-20 DEG C of Refrigerator stores are for subsequent use.
4. the preparation method of the ovomucoid rabbit polyclonal antibody according to any one of claims 1 to 3, is characterized in that: described antibody purification procedures, specifically comprises the steps:
1) by the ultrasonic 10-30 minute of all reagent required in experiment, bubble is got rid of;
2) purification column is balanced: first use flushing pipeline 1-2h, the reagent selected is the phosphoric acid buffer of pH 7.4, flow velocity 0.5-1.5mL/min; Get purification column after flushing of pipeline, and a part of ethanolic soln of Feng Zhuyong is slowly poured out, leave the ethanolic soln of post height 1/3rd; Purification column and protein purification instrument are connected, rinse balance pillar with the phosphoric acid buffer of pH 7.4, 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 0.5-1.5mL ovomucoid rabbit polyclonal antibody serum to be purified and phosphoric acid buffer equal-volume dilutes, flow velocity is set to 0.2-0.8mL/min, then by mixed solution upper prop, can demonstrate foreign protein peak in purifying instrument ultraviolet conductance figure;
4) wash-out: after there is foreign protein peak, continuation phosphoric acid buffer rinses pillar, stops after baseline reaches balance; With the glycine-HCI wash buffer pillar of pH 2.7, flow velocity 0.5-1.5mL/min, the immunoglobulin (Ig) be now combined with Protein A-Sepharose 4B is eluted;
5) collect: after adding elutriant, note the change of instrument ultraviolet conductance figure line, just start to collect elutriant 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 buffered saline buffer, flow velocity 1-8mL/min measures the pH value flowing out damping fluid simultaneously with pH test paper, until its display neutrality; Be finally that 20% ethanolic soln rinses purification column 20min by mass concentration, and make in pillar, to be full of 20% ethanolic soln, be sealed in 4 DEG C of refrigerators;
7) antibody is preserved: dialysed three days under 4 DEG C of environment by the ovomucoid antibody after purifying, dialyzate is the PBS solution of 0.01M, dialyzate changes three to four times every day, mixes, save backup after packing in-20 DEG C of refrigerators after taking out with isopyknic glycerol.
5. an immune analysis method for the ovomucoid rabbit 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 ovomucoid standardized solution, be coated in the enzyme 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 ovomucoid standardized solution, then adds 50 μ L ovomucoid rabbit polyclonal antibodies, and the extension rate of antibody is 1:40000; Competing 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 enzyme plate, 37 DEG C of baking oven reaction 30min, PBST washing lotions wash plate 4 times, and per minute once;
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 termination reactions;
7) reading: read absorbance by microplate reader, adopts 450-650nm dual wavelength mode;
8) drawing standard curve;
Preferably, the solution formula of above use 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 sodium acetate, anhydrous 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 Urea 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 100mg TMB and be dissolved in 10mL DMSO, 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.
6. immune analysis method according to claim 5, is characterized in that: step 1) in, the package amount of ovomucoid is 0.03 ~ 0.05 μ g/ hole; Preferably, 0.05 μ g/ hole.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105181954A (en) * | 2015-10-12 | 2015-12-23 | 天津科技大学 | Preparation method of rabbit anti ovotransferrin allergen polyclone antibody and immunoassay method thereof |
| CN106124766A (en) * | 2016-07-05 | 2016-11-16 | 天津师范大学 | Use the method for carbendazim content in carbendazim detection of specific antibody edible fungi |
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| CN106124766A (en) * | 2016-07-05 | 2016-11-16 | 天津师范大学 | Use the method for carbendazim content in carbendazim detection of specific antibody edible fungi |
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Application publication date: 20150930 |