CN110950952A - Method for producing antibody - Google Patents
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- CN110950952A CN110950952A CN201911302717.0A CN201911302717A CN110950952A CN 110950952 A CN110950952 A CN 110950952A CN 201911302717 A CN201911302717 A CN 201911302717A CN 110950952 A CN110950952 A CN 110950952A
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
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Abstract
The invention provides a method for preparing an antibody. The method comprises the following steps: subjecting a first animal to be immunized to active immunization with an antigen, so as to obtain a first serum containing antibodies; passively immunizing a second animal to be immunized with the first antibody-containing serum to obtain a second antibody-containing serum; the serum containing the secondary antibody is subjected to a purification treatment to obtain the secondary antibody. The coverage rate of the antibody obtained by the method is obviously improved.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a method for preparing an antibody.
Background
Antibodies (antibodies) are proteins that protect the body from antigenic stimuli. It (immunoglobulin is not just an antibody) is a large Y-shaped protein secreted by plasma cells (effector B cells), used by the immune system to identify and neutralize foreign substances such as bacteria, viruses, etc., and found only in body fluids such as blood of vertebrates, and the cell membrane surface of B cells thereof. Antibodies recognize a unique feature of a particular foreign object, referred to as an antigen.
An antigen is usually composed of multiple epitopes, one epitope stimulates the body, and the antibody produced by a B lymphocyte stimulated by the antigen is called monoclonal antibody (monoclonal antibody). Stimulating the body by a plurality of antigenic determinants to correspondingly generate various monoclonal antibodies, wherein the monoclonal antibodies are mixed together to form polyclonal antibodies, and the antibodies generated in the body are the polyclonal antibodies; in addition to the diversity of epitopes, the same epitope can stimulate the body to produce five classes of antibodies, IgG, IgM, IgA, IgE, and IgD. The antigen stimulates the body to produce an immunological reaction, and a group of globulins, which are immunoglobulins, synthesized and secreted by plasma cells of the body and have the ability of specifically binding to the antigen are antibodies.
For polyclonal antibodies, subcutaneous or intramuscular immunization, intradermal immunization, lymph node immunization, and mixed immunization are common examples. An animal (commonly used animals are rabbit, sheep, dog, etc.) is immunized with the antigen, antiserum is separated and the antibody is purified to obtain a polyclonal antibody.
The conventional method for preparing polyclonal antibody of host protein is to perform active immunization on animals (such as rabbit, goat and the like) for many times, collect serum, and purify to obtain the polyclonal antibody. However, the coverage rate of the polyclonal antibody of the host protein obtained by the prior art is low, and the requirement of monitoring the host cell residue in the biological medicine product in the biological medicine production is difficult to meet.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
conventionally, active immunization is realized by stimulating an antigen to enable an organism to generate an antibody, and is a common method for preparing the antibody; passive immunization refers to the passive reception of antibodies by the body, so that the body can obtain immunity, such as conventional vaccination. The inventors have surprisingly and unexpectedly found in experiments that if antibodies are prepared by a combination of active and passive immunization, the coverage of the obtained antibodies is significantly improved compared to the coverage of the antibodies obtained by active immunization alone.
In a first aspect of the invention, the invention proposes the use of passive immunization for the preparation of antibodies. As described above, the inventors have unexpectedly and surprisingly found in experiments that passive immunization is applied to the preparation of antibodies, i.e., antibodies are injected into an animal to be immunized, and then serum of the animal injected with the antibodies is collected, and found that the coverage rate of the antibodies in the collected serum is significantly improved compared to that of the original antibodies.
In a second aspect of the invention, the invention features a method of making an antibody. According to an embodiment of the invention, the method comprises: subjecting a first animal to be immunized to active immunization with an antigen, so as to obtain a first serum containing antibodies; passively immunizing a second animal to be immunized with the first antibody-containing serum to obtain a second antibody-containing serum; the serum containing the secondary antibody is subjected to a purification treatment to obtain the secondary antibody. According to the antibody preparation method provided by the embodiment of the invention, the coverage rate of the obtained antibody is obviously improved.
According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:
according to an embodiment of the invention, the method further comprises subjecting the first antibody-containing serum to a purification treatment so as to obtain first antibodies; mixing the first antibody with the second antibody so as to obtain the antibody. Further, the coverage of the antibody was further improved.
According to an embodiment of the invention, the antigens are cellular holoprotein HCPs.
According to the specific embodiment of the invention, the antigen is fermentation engineering bacteria HCPs.
According to a particular embodiment of the invention, the antigens are pichia pastoris HCPs.
According to a particular embodiment of the invention, the first and/or second animal to be immunized is a new zealand white rabbit.
According to the embodiment of the invention, the fermentation engineering bacteria HCPs are obtained by the following steps: carrying out fermentation treatment on fermentation engineering bacteria carrying empty carriers so as to enable OD600 of the fermentation engineering bacteria to reach at least 400, preferably not more than 600, taking fermentation liquor as immunogen 1 (comprising fermentation supernatant and thalli), taking the fermentation broth as immunogen 2, and taking the thalli of the fermentation engineering bacteria as immunogen 3; and (3) performing affinity chromatography treatment on the fermentation liquor, wherein the chromatography treatment liquor is used as immunogen 4. In the preparation process of the antibody, the inventor simulates the production process of engineering bacteria fermentation products and adopts fermentation engineering bacteria carrying no-load carriers to carry out fermentation so as to obtain the immunogen. The inventor finds that the coverage rate of the obtained antibody is further remarkably improved compared with the antibody obtained by taking any one of the four immunogens as the immunogen immune individual respectively after the fermentation process and then mixing the obtained antibodies.
According to a specific embodiment of the present invention, the affinity chromatography of the fermentation broth is performed as follows: coupling about 10-30 mg of polyclonal antibody against Pichia Pastoris cell whole protein HCPs containing pPIC9K blank vector by adopting Bogelong Ezfast protein ADiamond affinity chromatography column, then loading 1-1.5 mg of Pichia Pastoris cell whole protein HCPs containing pPIC9K blank vector, and collecting penetrating fluid, namely the required immunogen 4.
According to the embodiment of the present invention, the method further comprises mixing the immunogen 1, the immunogen 2, the immunogen 3 and the immunogen 4 with Freund's complete adjuvant in equal volume respectively so as to obtain an immunogen mixture 1, an immunogen mixture 2, an immunogen mixture 3 and an immunogen mixture 4. Freund's complete adjuvant is a water-in-oil emulsion containing cell wall components of Mycobacterium tuberculosis. Adjuvant activity results from the sustained release of immunogen in oil droplets and stimulates local immune response.
According to the embodiment of the present invention, the method further comprises mixing the immunogen 1, the immunogen 2, the immunogen 3 and the immunogen 4 with Freund's incomplete adjuvant in equal volume respectively, so as to obtain immunogen mixture A, immunogen mixture B, immunogen mixture C and immunogen mixture D. Freund's incomplete adjuvant does not contain Mycobacterium tuberculosis component.
According to an embodiment of the invention, the active immunization is performed by: respectively utilizing the immunogen mixed liquor 1, the immunogen mixed liquor 2, the immunogen mixed liquor 3 and the immunogen mixed liquor 4 to carry out initial immunization on four individuals of the first animal to be immunized; and (3) performing boosting immunity on the four individuals of the first animal to be immunized by using the immunogen mixed liquor A, the immunogen mixed liquor B, the immunogen mixed liquor C and the immunogen mixed liquor D every 7-10 days. The initial immunization is carried out on the animal by adopting the immunogen mixed liquor containing the Freund complete adjuvant, and the boosting immunization is carried out on the animal by adopting the immunogen mixed liquor containing the Freund incomplete adjuvant, so that the local immune reaction is effectively stimulated on one hand, and the side effect is effectively reduced on the other hand.
According to an embodiment of the present invention, before the passive immunization, the first antibody-containing serum is diluted. Specifically, the dilution treatment is performed by 2-4 times, and preferably by 3 times.
According to the present example, the sera obtained after the first and second boosters were combined to obtain a passive immunization serum a, a passive immunization serum B, a passive immunization serum C, and a passive immunization serum D. The inventors found that the coverage of antibodies can be further improved by using the sera obtained after the first and second boosters and combining them as the sera for the subsequent passive immunization.
According to an embodiment of the invention, the passive immunization is performed by: performing initial immunization on four individuals of the second animal to be immunized by using the serum A for passive immunization, the serum B for passive immunization, the serum C for passive immunization and the serum D for passive immunization respectively; and boosting four individuals of the second animal to be immunized by using the serum A for passive immunization, the serum B for passive immunization, the serum C for passive immunization and the serum D for passive immunization every 7-10 days.
According to an embodiment of the present invention, when the antibody titer in the serum reaches at least 50 ten thousand, preferably, not more than 400 ten thousand, the first antibody-containing serum and the second antibody-containing serum are separately subjected to a purification treatment.
According to an embodiment of the invention, the mass ratio of the antigen to the first animal subject to be immunized is 1: 1-3: 2(μ g/g).
According to an embodiment of the invention, the volume-to-mass ratio of the first antibody-containing serum to the second individual animal to be immunized is 1: 20-1: 90 (ml/kg).
In a third aspect of the invention, the invention features a polyclonal antibody. According to an embodiment of the present invention, the polyclonal antibody is obtained by the method described above. The polyclonal antibody according to the embodiment of the invention has a coverage rate which is remarkably improved compared with the polyclonal antibody obtained by adopting single active immunization.
Drawings
FIG. 1 is an antiserum titer of an active immunization group according to an embodiment of the present invention;
FIG. 2 is an antiserum titer of a passive immunization group according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of antibody purification according to the example of the present invention;
FIG. 4 shows the recognition results of the antibodies of the active and passive immunizations on the whole protein HCPs of Pichia Pastoris cells containing pPIC9K blank vector according to the embodiment of the present invention;
FIG. 5 is a graph showing the results of PDQuest software analyzing the coverage of antibodies prepared according to the present invention; and
FIG. 6 is a graph comparing 2D-western blot patterns of active versus mixed antibodies according to an embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Aiming at the defect that the conventional antibody preparation mode has low coverage rate on host protein antibodies, the inventor of the application finds a preparation method of the antibody with high coverage rate on host cell proteins.
According to the invention, the rabbit in New Zealand is immunized in a mode of combining active immunization and passive immunization, and the coverage rate of the rabbit polyclonal antibody is evaluated by using 2D electrophoresis and western blot technology, so that the coverage rate of the antibody is obviously improved.
EXAMPLE 1 preparation of Rabbit-derived polyclonal antibodies against Pichia Pastoris cell host protein containing pPIC9K blank vector
1) An experimental instrument: a mixed type ball mill manufacturer: german Leichi
Electronic balance manufacturers: mettler
2) Experimental materials: new zealand white rabbit manufacturer: qingdaokang Dabiol SCXK (Lu) female 2-2.5kg
PBS powder pack manufacturer: BOSTER
Freund complete adjuvant manufacturer: SIGMA
Freund incomplete adjuvant manufacturer: SIGMA
Immunogen: pichia Pastoris cell holoprotein HCPs1 (thalli + supernatant) containing pPIC9K blank vector, Pichia Pastoris cell fermentation supernatant HCPs2 containing pPIC9K blank vector, Pichia Pastoris cell thalli HCPs3 containing pPIC9K blank vector, and HCPs4 with low immunogenicity and captured by affinity chromatography.
The preparation process of the immunogen comprises the following steps: after a culture medium is prepared in a fermentation tank, disinfection and cooling are carried out, Pichia Pastoris cells containing pPIC9K blank carriers are inoculated into the fermentation tank, aeration and stirring are carried out, the temperature is adjusted to be 29 +/-3 ℃, fermentation is started, glycerol and supplementary urea are fed in the process, then the temperature is controlled to be 25-33 ℃ in stages according to time, and the expression speed of products is increased along with the supplement amount of methanol. After about 60 hours of fermentation, adding ammonium sulfate to supplement a nitrogen source, and continuing to ferment until the end. And (3) fermenting for about 90-130 hours, and collecting the sample when the OD600 value reaches more than 400 but not more than 600 to obtain the fermentation liquor. And (3) centrifuging the fermentation liquor, respectively obtaining fermentation supernatant and thalli, carrying out ultrasonic crushing treatment on the thalli at 4-10 ℃, and centrifuging to obtain thalli supernatant. Respectively taking the thallus supernatant and the fermentation supernatant, filtering the mixture by using a 3KD membrane package at the temperature of about 4-10 ℃, and taking the thallus HCPs and the fermentation supernatant HCPs with the density of more than 3KD for freeze-drying. Before immunization, respectively taking freeze-dried powder of HCPs of fermentation supernatant and freeze-dried powder of HCPs of thalli to dissolve by PBS buffer solution, thus obtaining the HCPs2 (immunogen 2) of immunogen fermentation supernatant and HCPs3 (immunogen 3) of thalli; mixing the obtained mixture in equal amount according to the protein content to obtain the required full protein HCPs1 (thallus + supernatant) (immunogen 1); HCPs4 (immunogen 4) of low immunogenicity captured by affinity chromatography.
The low immunogenicity of HCPs4 captured by affinity chromatography was obtained as follows:
coupling about 10-30 mg of polyclonal antibody against Pichia Pastoris cell whole protein HCPs containing pPIC9K blank vector by adopting Bogelong Ezfast protein A Diamond affinity chromatography column, then loading 1-1.5 mg of Pichia Pastoris cell whole protein HCPs containing pPIC9K blank vector, and collecting penetrating fluid, namely the required immunogen 4(HCPs 4).
3) The experimental process comprises the following steps:
preparing a primary immune solution: respectively taking an immunogen solution and Freund's complete adjuvant, mixing the immunogen solution and Freund's complete adjuvant in equal volume, and oscillating the mixture on a mixed ball mill to fully emulsify the mixture;
preparing a booster immune solution: and respectively taking the immunogen solution and Freund incomplete adjuvant, mixing the immunogen solution and Freund incomplete adjuvant in equal volume, and oscillating the mixture on a mixed ball mill to fully emulsify the mixture.
The immunization method comprises the following steps: adopting back multi-point injection method, namely selecting multiple points at two sides of rabbit spinal column to perform subcutaneous injection (1-2 ml blood is taken from ear vein before each immunization, and upper layer serum is taken and stored at-20 deg.C);
immunogen injection amount: about 500 μ g/kg;
animals to be immunized: 2-2.5kg of female New Zealand white rabbits;
removing air bubbles from the injector before injection, shaving hair at the injection site, sterilizing exposed skin with 75% ethanol, pinching out skin, inserting needle at 15 ° angle to skin, with the needle depth of 1-2cm, not penetrating into muscle, placing the needle at the injection site for several seconds, slightly pulling out, sterilizing at the injection site with 75% ethanol, and taking 3-4 injection points for each immunization.
<1> active immunization procedure:
active immunization of new zealand white rabbits was performed as per table 1: comprises one priming and three boosting.
Table 1:
<2> Passive immunization procedure:
mixing the sera obtained from the first and second boosters in the active immunization according to the corresponding rabbit group, filtering with 0.22 μm sterile filter head, packaging with 100 μ l/tube, and storing at-20 deg.C.
The serum is thawed before passive immunization, diluted to 300 μ l with physiological saline, injected into ear vein of rabbit group corresponding to immunization 5min before immunization, and then injected into subcutaneous rabbit, and 100 μ l of serum obtained by primary and secondary booster immunization is injected into each rabbit.
New Zealand white rabbits were passively immunized as per Table 2.
Table 2:
<3> titer monitoring of antisera:
the titers of the antisera after immunization were determined by indirect ELISA:
the antiserum (antibody-containing serum) titers of the active immunization groups are shown in FIG. 1. The antiserum (antibody-containing serum) titers of the passive immunization groups are shown in FIG. 2.
After the active immunization group and the passive immunization group are immunized for multiple times, the antiserum titer is over 50 ten thousand, and whole blood collection can be carried out.
<4> Whole blood Collection and antibody purification
Adopting an abdominal artery blood collection method, injecting a 3% sodium pentobarbital solution into ear vein of a white rabbit in New Zealand at a ratio of 1.2ml/kg for anesthesia before blood collection, cutting the abdomen with an operation, finding the abdominal artery, collecting whole blood, standing the obtained blood at 4 ℃ overnight, centrifuging at 4 ℃ and 3000rpm for 15min, and taking upper serum. Diluting the upper layer serum with 2 times volume of sodium acetate buffer solution (pH about 5.0), mixing, adjusting pH to 4.8 with 1M hydrochloric acid, placing on a magnetic stirrer, dripping n-octanoic acid in the process of slow stirring to make the final concentration be 3.3%, standing overnight at 4 deg.C, centrifuging the solution at 4 deg.C and 10000rpm for 20min the next day, taking supernatant solution, filtering, adding 1/10 volume of PBS buffer solution, mixing, adjusting pH to 7.4 with 1M sodium hydroxide solution, placing on a magnetic stirrer, adding 0.308g solid ammonium sulfate per ml solution in the process of slow stirring, stirring for 30min, standing overnight at 4 deg.C to make the antibody fully precipitate, centrifuging at 4 deg.C and 10000rpm for 20min, discarding supernatant, adding equal volume of PBS buffer solution to redissolve the precipitate, further purifying the antibody by Protein A affinity chromatography, collecting the elution peak and penetration peak respectively, the purification effect of the antibody was evaluated by SDS-PAGE, and the results are shown in FIG. 3 (non-reducing electrophoresis on the left side and reducing electrophoresis on the right side). It can be seen that the antibody with higher purity (i.e., the eluted group) was obtained after Protein A affinity chromatography.
EXAMPLE 2 examination of Rabbit-derived polyclonal antibodies against Pichia Pastoris cell host proteins containing pPIC9K blank vector
<1> the recognition ability of the prepared antibodies to Pichia pastoris cell whole protein HCPs containing pPIC9K blank vector was examined using 1-D electrophoresis-western blot, and the results are shown in FIG. 4.
From the results of 1-D electrophoresis-western blot mapping, the corresponding passive immune group has obviously increased recognition bands to Pichia Pastoris cell holoprotein HCPs containing pPIC9K blank vector, especially has more band parts under 50KD recognized than the active immune group, and the antibodies of the active immune group and the passive immune group are combined to be used as the antibody prepared by the invention.
<2> comparison of coverage rates of antibodies prepared by the method of the present invention and active immunoregulation antibodies on Pichia Pastoris cell holoprotein HCPs containing pPIC9K blank vector by 2-D electrophoresis-western blot
The 2-D electrophoresis-western blot of the antibody prepared by the method of the invention is shown in FIG. 5. Analyzing results according to PDQuest software: the statistical number of protein spots of the silver staining pattern of the whole protein HCPs of the Pichia Pastoris cells containing the pPIC9K blank vector is 602; the statistical number of spots of WB atlas of the self-made antibody is 552; the number of the common spots matched with the two maps is 258; the coverage rate of the self-made antibody is 552/(602+ 552-.
FIG. 6 is a 2D-Western Blot comparison of the active and mixed antibodies, and the results show that the 2D-Western Blot antibody-recognized protein spots are significantly increased after the passive immunization antibody is added.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (15)
1. Use of passive immunization for the preparation of an antibody.
2. A method of producing an antibody, comprising:
subjecting a first animal to be immunized to active immunization with an antigen, so as to obtain a first serum containing antibodies;
passively immunizing a second animal to be immunized with the first antibody-containing serum to obtain a second antibody-containing serum;
the serum containing the secondary antibody is subjected to a purification treatment to obtain the secondary antibody.
3. The method of claim 2, further comprising subjecting the first antibody-containing serum to a purification process to obtain first antibodies;
mixing the first antibody with the second antibody so as to obtain the antibody.
4. The method of claim 2, wherein the antigen is cellular whole protein HCPs, optionally the antigen is fermentation engineered bacteria HCPs, optionally the antigen is Pichia pastoris HCPs, optionally the first and/or second animal to be immunized is a New Zealand white rabbit.
5. The method of claim 4, wherein the fermentation engineering bacteria HCPs are obtained by:
fermenting the fermentation engineering bacteria carrying the empty carriers so as to enable the OD600 of the fermentation engineering bacteria to reach at least 400, preferably not more than 600;
taking fermentation liquor as immunogen 1, taking supernatant of the fermentation liquor as immunogen 2, and taking thalli of fermentation engineering bacteria as immunogen 3;
and (3) performing affinity chromatography treatment on the fermentation liquor, wherein the chromatography treatment liquor is used as immunogen 4.
6. The method of claim 5, further comprising mixing the immunogen 1, the immunogen 2, the immunogen 3 and the immunogen 4 with Freund's complete adjuvant in equal volumes to obtain immunogen mixture 1, immunogen mixture 2, immunogen mixture 3 and immunogen mixture 4.
7. The method of claim 5, further comprising mixing said immunogen 1, immunogen 2, immunogen 3, and immunogen 4 with an equal volume of Freund's incomplete adjuvant to obtain immunogen mixture A, immunogen mixture B, immunogen mixture C, and immunogen mixture D, respectively.
8. The method of claim 6 or 7, wherein the active immunization is carried out by:
respectively utilizing the immunogen mixed liquor 1, the immunogen mixed liquor 2, the immunogen mixed liquor 3 and the immunogen mixed liquor 4 to carry out initial immunization on four individuals of the first animal to be immunized;
and (3) performing boosting immunity on the four individuals of the first animal to be immunized by using the immunogen mixed liquor A, the immunogen mixed liquor B, the immunogen mixed liquor C and the immunogen mixed liquor D every 7-10 days.
9. The method of claim 2, wherein prior to passive immunization, the method further comprises diluting the first antibody-containing serum, optionally by a factor of 2-4, preferably 3.
10. The method according to claim 8, wherein the sera obtained after the first and second boosters are combined to obtain a passive immunization serum A, a passive immunization serum B, a passive immunization serum C, and a passive immunization serum D.
11. The method of claim 9 or 10, wherein the passive immunization is carried out by:
performing initial immunization on four individuals of the second animal to be immunized by using the serum A for passive immunization, the serum B for passive immunization, the serum C for passive immunization and the serum D for passive immunization respectively;
and boosting four individuals of the second animal to be immunized by using the serum A for passive immunization, the serum B for passive immunization, the serum C for passive immunization and the serum D for passive immunization every 7-10 days.
12. The method according to claim 2 or 3, wherein the first antibody-containing serum and the second antibody-containing serum are purified separately when the antibody titer in the serum reaches at least 50 ten thousand;
preferably, the antibody titer in the serum does not exceed 400 million.
13. The method according to claim 2, wherein the mass ratio of the antigen to the first animal subject to be immunized is 1: 1-3: 2.
14. The method according to claim 2, wherein the volume-to-mass ratio of the first antibody-containing serum to the second individual animal to be immunized is 1: 20-1: 90.
15. A polyclonal antibody obtained by the method according to any one of claims 2 to 14.
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| CN1186500A (en) * | 1995-04-19 | 1998-07-01 | 波利门科学生物免疫研究有限公司 | Monoclonal antibodies against HIV-1 and vaccines made thereof |
| CN102172400A (en) * | 2002-10-04 | 2011-09-07 | 生物发明国际公司 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
| CN102233133A (en) * | 2010-04-29 | 2011-11-09 | 上海市农业科学院 | Preparation method and application of Newcastle disease virus infected immune complex vaccines |
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| CN1186500A (en) * | 1995-04-19 | 1998-07-01 | 波利门科学生物免疫研究有限公司 | Monoclonal antibodies against HIV-1 and vaccines made thereof |
| CN102172400A (en) * | 2002-10-04 | 2011-09-07 | 生物发明国际公司 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
| CN102233133A (en) * | 2010-04-29 | 2011-11-09 | 上海市农业科学院 | Preparation method and application of Newcastle disease virus infected immune complex vaccines |
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Application publication date: 20200403 |