CN110724196A - Preparation method of anti-idiotype egg yolk antibody - Google Patents
Preparation method of anti-idiotype egg yolk antibody Download PDFInfo
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- CN110724196A CN110724196A CN201911133973.1A CN201911133973A CN110724196A CN 110724196 A CN110724196 A CN 110724196A CN 201911133973 A CN201911133973 A CN 201911133973A CN 110724196 A CN110724196 A CN 110724196A
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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Abstract
The invention discloses a preparation method of an anti-idiotype yolk antibody, which relates to the technical field of antibody preparation and is used for preparing immunogen; mixing the immunogen and incomplete Freund's adjuvant, uniformly stirring, and injecting an immune mouse subcutaneously according to a first preset condition to obtain an antibody of the mouse to the antigen; mixing the antibody of the mouse anti-antigen with incomplete Freund's adjuvant, injecting an immune layer chicken, and immunizing for a preset number of times according to a second preset condition; collecting the eggs laid by the laying hens on the same day; collecting high-immunity eggs according to a fourth preset condition; separating and preserving the yolk of the high-immunity egg and crushing a yolk tissue; after the yolk liquid is treated, collecting filtrate to obtain an anti-idiotype yolk antibody crude product; purifying the crude anti-idiotype yolk antibody; and respectively carrying out titer detection, specificity detection and competitive inhibition test on the anti-idiotype yolk antibody.
Description
Technical Field
The invention belongs to the technical field of antibody preparation, and particularly relates to a preparation method of an anti-idiotype egg yolk antibody.
Background
At present, the preparation method of anti-idiotype antibody is mostly anti-idiotype monoclonal antibody, the method firstly prepares monoclonal antibody of antigen, screens monoclonal antibody with neutralizing effect of immunity to antigen function, then immunizes mouse again with the monoclonal antibody to prepare anti-idiotype monoclonal antibody, wherein, the hybridoma cell strain is prepared twice through twice cell fusion, which wastes time and labor.
Moreover, it takes at least one year to prepare a useful anti-idiotype monoclonal antibody, and the development process is long and costly. Meanwhile, the prior art also has the preparation of anti-idiotype polyclonal antibodies, such as anti-idiotype rabbit antibodies, and the method is difficult to perform aseptic treatment when animals are killed when serum is taken, so that the method is not suitable for industrial production.
Disclosure of Invention
The embodiment of the application provides a preparation method of the anti-idiotype egg yolk antibody, so that the technical problems that an anti-idiotype monoclonal antibody in the prior art is long in research and development period and high in cost, the anti-idiotype rabbit antibody needs to kill a large number of organisms, sterile treatment is difficult to carry out, and industrial production is not suitable are solved, the research and development period and the production period are shortened, time and labor are saved, the cost is saved, the use approaches are various, a large number of organisms are not needed, the resource is rich, sterile treatment is easy to carry out, and the technical effect of industrial production can be achieved.
The embodiment of the invention provides a preparation method of an anti-idiotype yolk antibody, which comprises the following steps: step 1: preparing an immunogen; step 2: mixing the immunogen obtained in the step 1 with incomplete Freund's adjuvant, uniformly stirring, and injecting an immune mouse subcutaneously according to a first preset condition to obtain an antibody of the mouse to the antigen; and step 3: mixing the antibody of the mouse anti-antigen obtained in the step 2 with incomplete Freund's adjuvant, injecting the mixture into muscles to immunize the laying hens, and immunizing for a preset number of times according to a second preset condition; and 4, step 4: collecting eggs laid by the laying hens on the same day 3-5 days after the second immunization, detecting the titer of the anti-idiotype antibody by using an ELISA method, and judging whether the laying hens meet a third preset condition; and 5: when the laying hens meet the third preset condition, high immunity eggs are collected according to a fourth preset condition; step 6: separating and preserving the yolk of the high-immunity egg and crushing a yolk tissue; and 7: after the egg yolk liquid is treated, standing to enable the mixed protein to continue to precipitate, sucking a first supernatant by using a suction pipe after a first preset time, centrifuging a turbid part at the bottom, sucking a second supernatant again, mixing the first supernatant and the second supernatant, filtering, and collecting a filtrate to obtain an anti-idiotype egg yolk antibody crude product; and 8: purifying the crude anti-idiotype yolk antibody product obtained in step 7 according to cold alcohol precipitation; and step 9: and respectively carrying out titer detection, specificity detection and competitive inhibition test on the anti-idiotype yolk antibody.
Preferably, in the step 1, the method further includes: when the molecular weight of the immunogen meets a preset threshold value, coupling the immunogen and protein molecules to obtain an immunogen complex, and then immunizing; wherein the coupling process specifically comprises: dissolving 10mg of carrier protein into 1mL of PBS to obtain a first solution, dissolving 1mg of immunogen protein or peptide into 1mL of distilled water to obtain a second solution, mixing the first solution and the second solution, dropwise adding 1mL of 0.25% glutaraldehyde solution while stirring, continuously stirring for 5-10 minutes after dropwise adding, standing at room temperature for 2-3 hours, and obtaining the immunogen compound.
Preferably, in the step 2, the first preset condition is that: the immunogen content of each mouse is 5-10 mug, the immunization times are 3-4 times, and the interval time is 7-10 days.
Preferably, in the step 2, the method further includes: collecting tail venous blood of the mouse 3-5 days after the second immunization, centrifuging to obtain serum, detecting the titer of the antibody of the serum anti-antigen, and judging whether the detection result meets a fifth preset condition; and when the fifth preset condition is met, respectively bleeding 5-7 days after the third immunization, centrifuging to obtain serum, and subpackaging.
Preferably, in the step 3, the second preset condition is specifically: the injection amount is 0.5mL each time, the amount of the antibody containing the mouse anti-antigen is 20-40 mug, immunization is carried out once every 7-10 days, and the preset times are 3-4 times.
Preferably, in the step 6, the yolk separation, preservation and breaking of yolk tissue of the high-immunity eggs includes: cleaning the high-immunity eggs, soaking the high-immunity eggs in 70% alcohol for a second preset time, then sending the high-immunity eggs into a purification room for airing, and separating egg yolks manually or mechanically; crushing yolk tissue by a crusher or a high-pressure homogenizer.
Preferably, in the step 8, the crude anti-idiotype yolk antibody preparation is purified, comprising: adding cold alcohol into the anti-idiotype yolk antibody crude product to enable the concentration of the anti-idiotype yolk antibody crude product to reach 50% -60% of the volume of yolk liquid, stirring and centrifuging to obtain a first precipitate; adding sterile distilled water into the first precipitate to keep the volume of the first mixed solution consistent with that of the crude product of the anti-idiotype egg yolk antibody, adding cold alcohol to reach 25-30% of the volume of the first mixed solution, stirring, and centrifuging to obtain a second precipitate; adding sterile distilled water into the second precipitate to keep the volume of the second mixed solution consistent with that of the crude product of the anti-idiotype egg yolk antibody, adding cold alcohol to reach 20-25% of the volume of the second mixed solution, stirring, and centrifuging to obtain a third precipitate; and treating the third precipitate to obtain a refined product of the anti-idiotype yolk antibody.
Preferably, in the step 9, the titer detection of the anti-idiotype yolk antibody comprises: diluting rabbit anti-antigen antibody to 1:500 by using coating buffer solution, coating the diluted rabbit anti-antigen antibody into ELISA reaction small holes, wherein the adding amount of each hole is 100 mu L, standing the rabbit anti-antigen antibody for 1h at normal temperature, removing liquid in the holes, and washing the rabbit anti-antigen antibody by using PBS; performing 10-fold serial dilution on the anti-idiotype yolk antibody by PBS, respectively adding the diluted anti-idiotype yolk antibody into coating holes, standing at normal temperature for 30min, removing liquid in the holes, and then washing by PBS; adding an enzyme-labeled goat anti-IgY antibody with the addition of 100 mu L per hole, standing at normal temperature for 30min, removing liquid in the holes, washing with PBS, adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, and detecting the light absorption value with an enzyme-labeling instrument.
Preferably, in the step 9, the specific detection of the anti-idiotype yolk antibody comprises: respectively coating rabbit anti-antigen antibody and rabbit anti-other peptide antibody to ELISA reaction holes, wherein the dosage of each hole is 100 mu L, standing at normal temperature for 1h, removing liquid in the holes, and washing with PBS; adding the anti-idiotype yolk antibody, wherein the adding amount of each hole is 100 mu L, standing at normal temperature for 30min, removing liquid in the holes, and then washing with PBS; adding an enzyme-labeled goat anti-IgY antibody with the addition of 100 mu L per hole, standing at normal temperature for 30min, removing liquid in the holes, washing with PBS, adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, and detecting the light absorption value with an enzyme-labeling instrument.
Preferably, in step 9, the competitive inhibition assay for the anti-idiotype yolk antibody comprises: coating rabbit anti-antigen antibody to the small reaction holes, wherein the addition of each hole is 100 mu L, standing for 1h at normal temperature, removing liquid in the holes, and washing with PBS; adding 100 μ L of antigen into each well, containing 10 μ g of antigen per well, replacing antigen with PBS as control, adding 100 μ L of antigen, standing at room temperature for 30min, removing liquid in the well, and washing with PBS; adding the anti-idiotype yolk antibody, wherein the adding amount of each hole is 100 mu L, standing at normal temperature for 30min, removing liquid in the holes, adding the enzyme-labeled goat anti-IgY antibody, wherein the adding amount of each hole is 100 mu L, standing at normal temperature for 30min, removing liquid in the holes, and washing with PBS; adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, and adding a stop solution to obtain a detection result.
One or more technical solutions in the embodiments of the present invention at least have one or more of the following technical effects:
the embodiment of the invention provides a preparation method of an anti-idiotype yolk antibody, which comprises the following steps: step 1: preparing an immunogen; step 2: mixing the immunogen obtained in the step 1 with incomplete Freund's adjuvant, uniformly stirring, and injecting an immune mouse subcutaneously according to a first preset condition to obtain an antibody of the mouse to the antigen; and step 3: mixing the antibody of the mouse anti-antigen obtained in the step 2 with incomplete Freund's adjuvant, injecting the mixture into muscles to immunize the laying hens, and immunizing for a preset number of times according to a second preset condition; and 4, step 4: collecting eggs laid by the laying hens on the same day 3-5 days after the second immunization, detecting the titer of the anti-idiotype antibody by using an ELISA method, and judging whether the laying hens meet a third preset condition; and 5: when the laying hens meet the third preset condition, high immunity eggs are collected according to a fourth preset condition; step 6: separating and preserving the yolk of the high-immunity egg and crushing a yolk tissue; and 7: after the egg yolk liquid is treated, standing to enable the mixed protein to continue to precipitate, sucking a first supernatant by using a suction pipe after a first preset time, centrifuging a turbid part at the bottom, sucking a second supernatant again, mixing the first supernatant and the second supernatant, filtering, and collecting a filtrate to obtain an anti-idiotype egg yolk antibody crude product; and 8: purifying the crude anti-idiotype yolk antibody product obtained in step 7 according to cold alcohol precipitation; and step 9: the anti-idiotype egg yolk antibody is subjected to titer detection, specificity detection and competitive inhibition tests respectively, so that the technical problems that the anti-idiotype monoclonal antibody in the prior art is long in research and development period and high in cost, a large amount of anti-idiotype rabbit antibodies need to be killed, sterile treatment is difficult to carry out, and industrial production is not suitable are solved, the research and development period and the production period are shortened, time and labor are saved, the cost is saved, the use ways are various, a large amount of killing is not needed, only resources are rich, sterile treatment is easy to carry out, and the technical effect of industrial production can be achieved.
The foregoing description is only an overview of the technical solutions of the present invention, and the embodiments of the present invention are described below in order to make the technical means of the present invention more clearly understood and to make the above and other objects, features, and advantages of the present invention more clearly understandable.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing an anti-idiotype yolk antibody according to an embodiment of the present invention.
Detailed Description
The embodiment of the application provides a preparation method of the anti-idiotype egg yolk antibody, and solves the technical problems that the anti-idiotype monoclonal antibody in the prior art is long in research and development period and high in cost, a large amount of anti-idiotype rabbit antibodies need to be killed, aseptic processing is difficult to carry out, and industrial production is not suitable.
The technical scheme in the embodiment of the invention has the following general idea: the preparation method of the anti-idiotype yolk antibody provided by the embodiment of the invention comprises the following steps of 1: preparing an immunogen; step 2: mixing the immunogen obtained in the step 1 with incomplete Freund's adjuvant, uniformly stirring, and injecting an immune mouse subcutaneously according to a first preset condition to obtain an antibody of the mouse to the antigen; and step 3: mixing the antibody of the mouse anti-antigen obtained in the step 2 with incomplete Freund's adjuvant, injecting the mixture into muscles to immunize the laying hens, and immunizing for a preset number of times according to a second preset condition; and 4, step 4: collecting eggs laid by the laying hens on the same day 3-5 days after the second immunization, detecting the titer of the anti-idiotype antibody by using an ELISA method, and judging whether the laying hens meet a third preset condition; and 5: when the laying hens meet the third preset condition, high immunity eggs are collected according to a fourth preset condition; step 6: separating and preserving the yolk of the high-immunity egg and crushing a yolk tissue; and 7: after the egg yolk liquid is treated, standing to enable the mixed protein to continue to precipitate, sucking a first supernatant by using a suction pipe after a first preset time, centrifuging a turbid part at the bottom, sucking a second supernatant again, mixing the first supernatant and the second supernatant, filtering, and collecting a filtrate to obtain an anti-idiotype egg yolk antibody crude product; and 8: purifying the crude anti-idiotype yolk antibody product obtained in step 7 according to cold alcohol precipitation; and step 9: the anti-idiotype yolk antibody is subjected to titer detection, specificity detection and competitive inhibition tests respectively, so that the technical effects of shortening the research and development and production periods, saving time and labor, saving cost, being various in use ways, free of killing a large amount of organisms, rich in resources, easy to perform sterile treatment and capable of realizing industrial production are achieved.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
Referring to FIG. 1, the method for preparing an anti-idiotype yolk antibody comprises:
step 1: and (3) preparing immunogen.
Further, in the step 1, the method further includes: when the molecular weight of the immunogen meets a preset threshold value, coupling the immunogen and protein molecules to obtain an immunogen complex, and then immunizing; wherein the coupling process specifically comprises: dissolving 10mg of carrier protein into 1mL of PBS to obtain a first solution, dissolving 1mg of immunogen protein or peptide into 1mL of distilled water to obtain a second solution, mixing the first solution and the second solution, dropwise adding 1mL of 0.25% glutaraldehyde solution while stirring, continuously stirring for 5-10 minutes after dropwise adding, standing at room temperature for 2-3 hours, and obtaining the immunogen compound.
Specifically, when the immunogen is prepared from a microorganism such as bacteria or viruses, the preparation of the immunogen is not required. If the molecular weight of the immunogen meets the requirement of a preset threshold, the immunogen is coupled with a protein molecule with the large molecular weight to prepare an immunogen complex, and then the immunogen is immunized. Further, in this embodiment, it is preferable that the predetermined threshold is required to be less than ten thousand, that is, when the molecular weight of the immunogen is less than ten thousand, it is necessary to couple the immunogen with a protein molecule having a large molecular weight to prepare an immunogen complex. The specific coupling method is as follows: 10mg of carrier protein (preferably bovine serum albumin)) Dissolving into 1mL of PBS (pH is 5.0-7.0), dissolving 1mg of immunogen protein or peptide into 1mL of distilled water, mixing and stirring the two, dropwise adding 1mL of 0.25% glutaraldehyde solution while stirring, continuously stirring for 5-10 minutes after dropwise adding, and then standing at room temperature to allow the mixture to continuously react for 2-3 hours to obtain the immunogen compound of the antigen. Subpackaging and storing at-20 deg.C for use. Among them, PBS is phosphate buffer saline (phosphate buffer saline), and generally serves as a solvent to dissolve the protective agent. It is a buffer solution which is most widely used in biochemical research, and the main component of the buffer solution is Na2HPO4、KH2PO4NaCl and KCl.
Step 2: mixing the immunogen obtained in the step 1 with incomplete Freund's adjuvant, uniformly stirring, and injecting the mixture subcutaneously for immunizing a mouse according to a first preset condition to obtain the antibody of the mouse for resisting the antigen.
Further, in the step 2, the first preset condition is that: the immunogen content of each mouse is 5-10 mug, the immunization times are 3-4 times, and the interval time is 7-10 days.
Further, in the step 2, the method further includes: collecting tail venous blood of the mouse 3-5 days after the second immunization, centrifuging to obtain serum, detecting the titer of the antibody of the serum anti-antigen, and judging whether the detection result meets a fifth preset condition; and when the fifth preset condition is met, respectively bleeding 5-7 days after the third immunization, centrifuging to obtain serum, and subpackaging.
Specifically, in the preparation of antibody (Ab1), an immunization process is first performed. The specific method comprises the following steps: mixing the immunogen or the immunogen complex obtained in the step 1 with incomplete Freund's adjuvant, and adopting equal volume mixing. The method comprises the steps of mixing an antigen or an immunogen compound with the same volume and incomplete Freund's adjuvant, uniformly stirring, and respectively injecting subcutaneously an immunized mouse, a rat and a rabbit, wherein each immunized immunogen of the mouse is 5-10 mu g, each immunogen of the rat is 10-20 mu g, each immunogen of the rabbit is 20-40 mu g, and the immunization is carried out 3-4 times at intervals of 7-10 days.
Further, when the immunization process is completed, the detection of the antibody titer is then required. The specific method comprises the following steps: and 3-5 days after the second immunization, respectively collecting tail venous blood of the mouse, tail venous blood of the rat and auricular venous blood of the rabbit, and centrifuging to obtain serum. And respectively detecting the titer of the anti-antigen antibody in the serum by using an ELISA method, and then judging whether the detection result meets a fifth preset condition. Wherein the fifth preset condition is that the titer reaches 1 × 10-4The above. If the titer reaches 1 × 10-4And (3) respectively bleeding 5-7 days after the third immunization, and centrifuging to obtain serum (blood is obtained by head breaking after anesthesia of mice and rats, and blood is obtained by carotid artery intubation after anesthesia of rabbits). Subpackaging the serum, and storing at-20 deg.C for use. Among them, ELISA is a shorthand of Enzyme Linked Immuno Sorbent Assay (ELISA), and refers to a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid phase carrier such as polystyrene, and immunoreaction is performed by using specific binding of antigen and antibody.
And step 3: and (3) mixing the mouse anti-antigen antibody obtained in the step (2) with incomplete Freund's adjuvant, injecting the mixture into an intramuscular injection layer, and immunizing the layer for a preset number of times according to a second preset condition.
Further, in the step 3, the second preset condition is specifically that: the injection amount is 0.5mL each time, the amount of the antibody containing the mouse anti-antigen is 20-40 mug, immunization is carried out once every 7-10 days, and the preset times are 3-4 times.
In particular, in the preparation of anti-idiotype yolk antibodies, it is first necessary to carry out an immunization process, in particular: after a mouse anti-antigen antibody (Ab1) and incomplete Freund's adjuvant are mixed in equal volume, the laying hens are immunized by intramuscular injection, the injection amount is 0.5mL each time, and the Ab1 content is 20-40 mu g. The immunization is carried out once every 7-10 days for 3-4 times.
And 4, step 4: collecting the eggs laid by the laying hens on the same day 3-5 days after the second immunization, detecting the titer of the anti-idiotype antibody by using an ELISA method, and judging whether the laying hens meet a third preset condition.
Specifically, when screening hyperimmune chickens, the method comprises the following specific steps: second immunizationAnd collecting the eggs laid by the laying hens on the same day 3-5 days later, detecting the titer of the anti-idiotype antibody by an ELISA method, and then judging whether the laying hens meet a third preset condition. Wherein the third predetermined condition is that the anti-idiotype antibody titer reaches 1X 10-4The above. If the titer of the anti-idiotype antibody is set to 1X 10-4In the above, the laying hen is selected as a high immunity chicken and is listed as an egg collecting object. No anti-idiotype antibody could be detected or the titer was not 1X 10-4When in use, the eggs can be sold according to the conventional method. Further, the criteria for the determination of anti-idiotype antibodies are: can react with the antibody of the antigen produced by different ethnic groups; this immune response can be suppressed by antigen competition; can induce the heterogeneous animal to produce antigen specific antibody.
And 5: and when the laying hens meet the third preset condition, acquiring high immunity eggs according to a fourth preset condition.
Specifically, after the high-immunity chicken is screened out, the high-immunity eggs are continuously collected, and the specific method comprises the following steps: the fourth preset condition is specifically as follows: collecting eggs of the hyperimmune chickens on the third day after the third immunization, continuously collecting for 20 days, performing additional immunization once after 20 days if necessary, and continuously collecting for 20 days until the laying hens are eliminated.
Step 6: and carrying out yolk separation and preservation and crushing of yolk tissues on the high-immunity eggs.
Further, in the step 6, the yolk separation, preservation and breaking of yolk tissues of the high-immunity eggs comprises: cleaning the high-immunity eggs, soaking the high-immunity eggs in 70% alcohol for a second preset time, then sending the high-immunity eggs into a purification room for airing, and separating egg yolks manually or mechanically; crushing yolk tissue by a crusher or a high-pressure homogenizer.
Specifically, the separation and preservation of the yolk specifically comprises the following steps: cleaning the collected eggs with tap water, soaking the eggs in 70% alcohol for a second preset time, wherein the second preset time is 10 minutes, sterilizing, taking out the eggs, then conveying the eggs into a purification room to dry the eggs, then separating the egg yolks by adopting a manual or machine, and storing the separated egg yolks for 1-6 months at-20 ℃ if the egg yolk antibodies are not prepared immediately. Further, the yolk tissue was mainly crushed by a crusher or a high-pressure homogenizer (60 Pa).
And 7: and (3) after the egg yolk liquid is treated, standing to enable the mixed protein to continue to precipitate, sucking a first supernatant by using a suction pipe after a first preset time, centrifuging the turbid part at the bottom, sucking a second supernatant again, mixing the first supernatant and the second supernatant, filtering, and collecting a filtrate to obtain the crude product of the anti-idiotype egg yolk antibody.
Specifically, after the egg yolk tissue is broken, the hybrid protein is removed, and the method comprises the following steps: removing impure protein by using a water dilution and acidification method, adding 10-40 times of sterile distilled water, uniformly stirring, then adjusting the pH value of the egg yolk liquid to 5.2-6.0 by using hydrochloric acid and sodium hydroxide solution to enable the egg yolk liquid to generate turbid precipitate, and placing the egg yolk liquid in a refrigerator at the temperature of 4-8 ℃ overnight to enable impure protein to continue to precipitate. And after the first preset time, wherein the first preset time is 24 hours, sucking the supernatant by using a suction pipe the next day, centrifuging the turbid part at the bottom for 20-40 minutes at the rotating speed of 3500-4000 rpm, and taking the supernatant. Mixing the two supernatants, filtering with microporous filter (pore size of 0.22 μm) to remove lipoprotein and fat, and collecting filtrate which is crude product of anti-idiotype yolk antibody.
And 8: purifying the crude anti-idiotype yolk antibody obtained in step 7 according to cold alcohol precipitation.
Further, in the step 8, the crude anti-idiotype yolk antibody preparation is purified, comprising: adding cold alcohol into the anti-idiotype yolk antibody crude product to enable the concentration of the anti-idiotype yolk antibody crude product to reach 50% -60% of the volume of yolk liquid, stirring and centrifuging to obtain a first precipitate; adding sterile distilled water into the first precipitate to keep the volume of the first mixed solution consistent with that of the crude product of the anti-idiotype egg yolk antibody, adding cold alcohol to reach 25-30% of the volume of the first mixed solution, stirring, and centrifuging to obtain a second precipitate; adding sterile distilled water into the second precipitate to keep the volume of the second mixed solution consistent with that of the crude product of the anti-idiotype egg yolk antibody, adding cold alcohol to reach 20-25% of the volume of the second mixed solution, stirring, and centrifuging to obtain a third precipitate; and treating the third precipitate to obtain a refined product of the anti-idiotype yolk antibody.
Specifically, after obtaining the crude anti-idiotype yolk antibody, the purification of the anti-idiotype yolk antibody is continued by the following specific operations: the crude anti-idiotype yolk antibody is purified by salt precipitation or cold alcohol precipitation, preferably cold alcohol precipitation in this embodiment. Firstly, adding cold alcohol with the temperature of-20 ℃ into the crude anti-idiotype yolk antibody product liquid for the first time to ensure that the concentration (volume ratio) of the cold alcohol reaches 50-60 percent of the volume of yolk liquid. Stirring thoroughly to make it turbid and precipitate. Centrifuging at 4000rpm for 20-30 minutes, and taking out the precipitate. Then, sterile distilled water is added to restore the volume of the crude product of the anti-idiotype yolk antibody, cold alcohol is added to reach 25 to 30 percent of the volume of the anti-idiotype yolk antibody liquid, and the mixture is fully stirred to generate turbid precipitation. Centrifuging at 4000rpm for 25-30 minutes, and taking out the precipitate. Then, sterile distilled water was continuously added to restore the volume of the crude anti-idiotype yolk antibody preparation. Then adding cold alcohol to reach 20-25% of the volume of the anti-idiotype yolk antibody solution, and fully stirring to ensure that turbid precipitation occurs. Centrifuging at 4000rpm for 20-30 minutes, and taking out the precipitate. Finally, the precipitate is dried in a constant temperature oven at 37 ℃ or dissolved by adding a proper amount of sterile distilled water, and a refined product of the solid or liquid anti-idiotype yolk antibody can be obtained.
And step 9: and respectively carrying out titer detection, specificity detection and competitive inhibition test on the anti-idiotype yolk antibody.
Specifically, the detection of the anti-idiotype yolk antibody mainly comprises titer detection, specificity detection and competitive inhibition test.
The titer of the yolk antibody is detected by adopting an indirect ELISA method. The specific procedures are as follows: diluting rabbit anti-antigen antibody to 1:500 with coating buffer solution, coating into ELISA reaction wells with each well being 100 μ L, standing at room temperature for 1h, discarding the liquid in the wells, washing with PBS for three timesAnd the time of each washing is 3min, performing 10-time serial dilution on the anti-idiotype egg yolk antibody by PBS, diluting 10 stages in total, respectively adding the diluted 10 stages into the coated holes, adding two repeated holes into each stage, adding PBS into a control hole, keeping the solution in each hole for 100 mu L, standing for 30min at normal temperature, discarding the liquid in the hole, and washing for three times by PBS, wherein the time of each washing is 3 min. Adding 100 μ L of enzyme labeled goat anti-IgY antibody (1:1000) per well, standing at room temperature for 30min, discarding the liquid in the well, washing with PBS for three times, each time for 3 min. Adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, and measuring the light absorption value (A) by using an enzyme-linked immunosorbent assay (ELIASA) at 492 nm. The A value of the sample to be detected is more than or equal to 2.1 times of the control hole, and the positive reaction is obtained. As shown in Table 1, the results of the titer test were obtained. As can be seen from Table 1, the anti-idiotype yolk antibody has a titer of 1X 10-5。
TABLE 1 yolk Ab2 potency test results
The specificity of the yolk Ab2 is detected by an indirect ELISA method, and the specific program is as follows: coating ELISA reaction pores with rabbit anti-antigen antibody and rabbit anti-other peptide antibody (1:500), respectively, standing at room temperature for 1h, discarding the liquid in the pores, washing with PBS for three times, each washing time being 3min, adding yolk anti-idiotype antibody (1:1000), standing at room temperature for 30min, discarding the liquid in the pores, washing with PBS for three times, each washing time being 3min, adding enzyme labeled goat anti-IgY antibody (1:1000), each pore 100 μ L, standing at room temperature for 30min, discarding the liquid in the pores, washing with PBS for three times, each washing time being 3 min. Adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, and measuring the light absorption value (A) by using an enzyme-linked immunosorbent assay (ELIASA) at 492 nm. The A value of the sample to be detected is more than or equal to 2.1 times of the control hole, and the positive reaction is obtained. As shown in Table 2, the results of the specific detection were obtained. As can be seen from Table 2, the anti-idiotype yolk antibody immunoreacts only with rabbit anti-antigen antibodies and not with rabbit anti-other antigen antibodies.
TABLE 2 yolk Ab2 specificity test result table
The specific operation method of the competitive inhibition test is as follows: coating reaction pores with rabbit anti-antigen antibody Ab1(1:1000), standing for 1h at normal temperature with 100 μ L per pore, removing liquid in the pores, washing with PBS for three times, each time washing for 3min, adding antigen with 100 μ L per pore, containing 10 μ g of antigen per pore, standing for 30min at normal temperature, removing liquid in the pores, washing with PBS for three times, adding anti-idiotype yolk antibody (1:1000), each pore with 100 μ L per pore, standing for 30min at normal temperature, removing liquid in the pores, adding enzyme labeled goat anti-IgY antibody (1:1000), each pore with 100 μ L, standing for 30min at normal temperature, removing liquid in the pores, washing with PBS for three times, each time washing for 3 min. Adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, replacing an antigen with PBS (phosphate buffer solution) as a control, detecting two groups of ELISA reaction A values, and calculating the significance of the mean value, the standard deviation and the difference, wherein the significance of the two groups of differences is required to be significant. It is shown that the yolk Ab2 can generate an immune reaction with the antigen-specific antibody of the xenogeneic animal, and the immune reaction can be inhibited by antigen competition. When the above conditions are met, it is shown that the yolk Ab2 is a yolk Ab2 with high titer, high specificity and intra-antigen image, and can be used as an antigen or a vaccine for the antigen.
Therefore, compared with the preparation method of the anti-idiotype yolk antibody, the preparation method of the anti-idiotype yolk antibody in the embodiment has the advantages of shorter development and production period, time and labor conservation, cost conservation and more diverse use ways. Compared with the anti-idiotype rabbit antibody, the method does not need to kill a large amount of organisms, only collects the eggs of the high-immunity chickens, has rich resources, is easy to carry out aseptic treatment, can be industrially produced, has various use ways and has lower cost. Furthermore, the anti-idiotype antibodies of the present embodiment mimic the function of an antigen, and can be used as an antigen or as a vaccine for an antigen, which can be used to treat and prevent diseases.
Furthermore, it is an object of the present invention to enable the rapid and simple preparation of anti-idiotype antibodies. The anti-idiotype antibody has the advantages of rich resources, simple preparation process, high yield, low cost, more various application ways, more convenient use and large-scale industrial production. Meanwhile, the anti-idiotype egg yolk antibody has stable performance, rich resources and low cost, the anti-idiotype egg yolk antibody has no ethnic boundary, is more convenient to popularize and apply, has more various using ways, is effective in injection, oral administration and soaking (for fish), and is more convenient and feasible to use.
One or more technical solutions in the embodiments of the present invention at least have one or more of the following technical effects:
the embodiment of the invention provides a preparation method of an anti-idiotype yolk antibody, which comprises the following steps: step 1: preparing an immunogen; step 2: mixing the immunogen obtained in the step 1 with incomplete Freund's adjuvant, uniformly stirring, and injecting an immune mouse subcutaneously according to a first preset condition to obtain an antibody of the mouse to the antigen; and step 3: mixing the antibody of the mouse anti-antigen obtained in the step 2 with incomplete Freund's adjuvant, injecting the mixture into muscles to immunize the laying hens, and immunizing for a preset number of times according to a second preset condition; and 4, step 4: collecting eggs laid by the laying hens on the same day 3-5 days after the second immunization, detecting the titer of the anti-idiotype antibody by using an ELISA method, and judging whether the laying hens meet a third preset condition; and 5: when the laying hens meet the third preset condition, high immunity eggs are collected according to a fourth preset condition; step 6: separating and preserving the yolk of the high-immunity egg and crushing a yolk tissue; and 7: after the egg yolk liquid is treated, standing to enable the mixed protein to continue to precipitate, sucking a first supernatant by using a suction pipe after a first preset time, centrifuging a turbid part at the bottom, sucking a second supernatant again, mixing the first supernatant and the second supernatant, filtering, and collecting a filtrate to obtain an anti-idiotype egg yolk antibody crude product; and 8: purifying the crude anti-idiotype yolk antibody product obtained in step 7 according to cold alcohol precipitation; and step 9: the anti-idiotype egg yolk antibody is subjected to titer detection, specificity detection and competitive inhibition tests respectively, so that the technical problems that the anti-idiotype monoclonal antibody in the prior art is long in research and development period and high in cost, a large amount of anti-idiotype rabbit antibodies need to be killed, sterile treatment is difficult to carry out, and industrial production is not suitable are solved, the research and development period and the production period are shortened, time and labor are saved, the cost is saved, the use ways are various, a large amount of killing is not needed, only resources are rich, sterile treatment is easy to carry out, and the technical effect of industrial production can be achieved.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to encompass such modifications and variations.
Claims (10)
1. A method for preparing an anti-idiotype egg yolk antibody, comprising:
step 1: preparing an immunogen;
step 2: mixing the immunogen obtained in the step 1 with incomplete Freund's adjuvant, uniformly stirring, and injecting an immune mouse subcutaneously according to a first preset condition to obtain an antibody of the mouse to the antigen;
and step 3: mixing the antibody of the mouse anti-antigen obtained in the step 2 with incomplete Freund's adjuvant, injecting the mixture into muscles to immunize the laying hens, and immunizing for a preset number of times according to a second preset condition;
and 4, step 4: collecting eggs laid by the laying hens on the same day 3-5 days after the second immunization, detecting the titer of the anti-idiotype antibody by using an ELISA method, and judging whether the laying hens meet a third preset condition;
and 5: when the laying hens meet the third preset condition, high immunity eggs are collected according to a fourth preset condition;
step 6: separating and preserving the yolk of the high-immunity egg and crushing a yolk tissue;
and 7: after the egg yolk liquid is treated, standing to enable the mixed protein to continue to precipitate, sucking a first supernatant by using a suction pipe after a first preset time, centrifuging a turbid part at the bottom, sucking a second supernatant again, mixing the first supernatant and the second supernatant, filtering, and collecting a filtrate to obtain an anti-idiotype egg yolk antibody crude product;
and 8: purifying the crude anti-idiotype yolk antibody product obtained in step 7 according to cold alcohol precipitation;
and step 9: and respectively carrying out titer detection, specificity detection and competitive inhibition test on the anti-idiotype yolk antibody.
2. The method of producing an anti-idiotype yolk antibody according to claim 1, further comprising, in step 1:
when the molecular weight of the immunogen meets a preset threshold value, coupling the immunogen and protein molecules to obtain an immunogen complex, and then immunizing;
wherein the coupling process specifically comprises: dissolving 10mg of carrier protein into 1mL of PBS to obtain a first solution, dissolving 1mg of immunogen protein or peptide into 1mL of distilled water to obtain a second solution, mixing the first solution and the second solution, dropwise adding 1mL of 0.25% glutaraldehyde solution while stirring, continuously stirring for 5-10 minutes after dropwise adding, standing at room temperature for 2-3 hours, and obtaining the immunogen compound.
3. The method for producing an anti-idiotype yolk antibody according to claim 1, wherein the first predetermined condition in step 2 is:
the immunogen content of each mouse is 5-10 mug, the immunization times are 3-4 times, and the interval time is 7-10 days.
4. The method of producing an anti-idiotype yolk antibody according to claim 1, wherein the step 2 further comprises:
collecting tail venous blood of the mouse 3-5 days after the second immunization, centrifuging to obtain serum, detecting the titer of the antibody of the serum anti-antigen, and judging whether the detection result meets a fifth preset condition;
and when the fifth preset condition is met, respectively bleeding 5-7 days after the third immunization, centrifuging to obtain serum, and subpackaging.
5. The method for producing an anti-idiotype yolk antibody according to claim 1, wherein in said step 3, said second predetermined condition is specifically:
the injection amount is 0.5mL each time, the amount of the antibody containing the mouse anti-antigen is 20-40 mug, immunization is carried out once every 7-10 days, and the preset times are 3-4 times.
6. The method of preparing anti-idiotype yolk antibody according to claim 1, wherein the step 6 of subjecting the hyperimmune egg to yolk separation, storage and disruption of yolk tissue comprises:
cleaning the high-immunity eggs, soaking the high-immunity eggs in 70% alcohol for a second preset time, then sending the high-immunity eggs into a purification room for airing, and separating egg yolks manually or mechanically;
crushing yolk tissue by a crusher or a high-pressure homogenizer.
7. The method of claim 1, wherein in step 8, the purification of the crude anti-idiotype yolk antibody comprises:
adding cold alcohol into the anti-idiotype yolk antibody crude product to enable the concentration of the anti-idiotype yolk antibody crude product to reach 50% -60% of the volume of yolk liquid, stirring and centrifuging to obtain a first precipitate;
adding sterile distilled water into the first precipitate to keep the volume of the first mixed solution consistent with that of the crude product of the anti-idiotype egg yolk antibody, adding cold alcohol to reach 25-30% of the volume of the first mixed solution, stirring, and centrifuging to obtain a second precipitate;
adding sterile distilled water into the second precipitate to keep the volume of the second mixed solution consistent with that of the crude product of the anti-idiotype egg yolk antibody, adding cold alcohol to reach 20-25% of the volume of the second mixed solution, stirring, and centrifuging to obtain a third precipitate;
and treating the third precipitate to obtain a refined product of the anti-idiotype yolk antibody.
8. The method of claim 1, wherein said step 9 of measuring the potency of said anti-idiotype yolk antibody comprises:
diluting rabbit anti-antigen antibody to 1:500 by using coating buffer solution, coating the diluted rabbit anti-antigen antibody into ELISA reaction small holes, wherein the adding amount of each hole is 100 mu L, standing the rabbit anti-antigen antibody for 1h at normal temperature, removing liquid in the holes, and washing the rabbit anti-antigen antibody by using PBS;
performing 10-fold serial dilution on the anti-idiotype yolk antibody by PBS, respectively adding the diluted anti-idiotype yolk antibody into coating holes, standing at normal temperature for 30min, removing liquid in the holes, and then washing by PBS;
adding an enzyme-labeled goat anti-IgY antibody with the addition of 100 mu L per hole, standing at normal temperature for 30min, removing liquid in the holes, washing with PBS, adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, and detecting the light absorption value with an enzyme-labeling instrument.
9. The method of claim 1, wherein said specific detection of said anti-idiotype yolk antibody in step 9 comprises:
respectively coating rabbit anti-antigen antibody and rabbit anti-other peptide antibody to ELISA reaction holes, wherein the dosage of each hole is 100 mu L, standing at normal temperature for 1h, removing liquid in the holes, and washing with PBS;
adding the anti-idiotype yolk antibody, wherein the adding amount of each hole is 100 mu L, standing at normal temperature for 30min, removing liquid in the holes, and then washing with PBS;
adding an enzyme-labeled goat anti-IgY antibody with the addition of 100 mu L per hole, standing at normal temperature for 30min, removing liquid in the holes, washing with PBS, adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, adding a stop solution, and detecting the light absorption value with an enzyme-labeling instrument.
10. The method of claim 1, wherein said step 9 of performing a competitive inhibition assay on said anti-idiotype yolk antibody comprises:
coating rabbit anti-antigen antibody to the small reaction holes, wherein the addition of each hole is 100 mu L, standing for 1h at normal temperature, removing liquid in the holes, and washing with PBS;
adding antigen with an amount of 100 μ L per well and containing 10 μ g of antigen per well, standing at room temperature for 30min, removing liquid in the well, and washing with PBS;
adding the anti-idiotype yolk antibody, wherein the adding amount of each hole is 100 mu L, standing at normal temperature for 30min, removing liquid in the holes, adding the enzyme-labeled goat anti-IgY antibody, wherein the adding amount of each hole is 100 mu L, standing at normal temperature for 30min, removing liquid in the holes, and washing with PBS;
adding a reaction substrate, developing for 10-15 min at normal temperature in a dark place, and adding a stop solution to obtain a detection result.
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