JP3747322B2 - A method of selectively separating antibody isoforms from egg yolk. - Google Patents

Description

Field of the Invention The present invention relates to a method of separation and purification rapid egg yolk, is particularly birds (hereinafter Evian) LGY (? Fc) separation and purification of antibodies from egg yolks of, and to yolk antibody obtained as a result. More specifically, the present invention relates to an absorption chromatography method for the purpose of suitable separation of an aqueous phase and a lipid phase using an insoluble-uncharged absorber, and a salting-out method for differential precipitation of egg yolk antibodies. Relates to a method for separating and purifying egg yolk antibodies from Evian egg yolk. The present invention also relates to the use of said egg yolk antibody for quantitative or qualitative immunoassay, or for the preparation of pharmaceutical ingredients for related pathogens.

Antibodies are widely used in many biological research and clinical topical drugs. Plasma obtained from over-immunized mammals (Sera) is the most common source of polyclonal antibodies. Antibodies obtained from such immune plasma belong to a group of proteins called “immunoglobulins”, of which immunoglobulin G (lgG) is the most abundant. The lgG molecule is composed of three domains, two Fab regions and one Fc region. The Fab portion is primarily involved in antigen binding. Although the Fc portion does not have the ability to bind antigen, it serves to manage several biological activities such as complement repair and Fc receptor organ binding.

In the field of immunodiagnosis, complete lgG molecules are not suitable for use in detection systems and immunoassays involving plasma from mammals. This is because the Fc region of the lgG molecule is capable of binding to Fc receptor organs, activates the mammalian system, and responds to rheumatoid factor in plasma obtained from mammals. Removal of the Fc region of the lgG molecule will lead to a reduction in frequently disturbing factors. (E. Lamoyi, Methods in Enzymology 121: 652-663 (1986)).

Some suggested uses of antibodies in immunotherapeutics include the treatment of patients affected by bacterial toxin poisoning or snake venom (see, eg, US Pat. Nos. 5,340,923 and 5,601,823), and piglets Newborns are protected from fatal intestinal colibacilosis (eg H. Brussow et al., J. Clin. Microbiol. 25: 982 (1987) and C. O. Tacket et al., New. Eng., J. Med. 318: 1240 (1998)). The Fc region of an antibody molecule is known as the most antigenic part of an immunoglobulin (EM Akita et al., J. Immunol. Methods. 162: 155-164 (1993)). (Ab ′) ₂ fragments are formed. Its F (ab ′) ₂ formation significantly reduces the number of sites that can be attributed to immunoglobulin allergies and is thus beneficial to humans or animals given immunoglobulins.

Recently, F (ab ′) ₂ antibody fragments of bivalent chromosomes have been more effective in immunodiagnostic tests (M. Muratsugu et al., J. Colloid Interface Sci 147: 378 (1991); L. Ortega-Vinuesa et al., J. Immunol Methods 90:29 (1996), and has proven to be more suitable than maternal lgG for the generation of immunoanalytes, including mammalian plasma.

However, F (ab ′) ₂ antibody fragments have not been widely used in clinical immunodiagnostic sets, contrary to expectations. This is made commercially from pepsin digestion of lgG and subsequent purification by chromatographic methods, and is likely due to the difficulty and cost effectiveness of large production of F (ab ′) ₂ fragments.

Ducks, the phylogenetic approximation of ducks, and reptiles such as turtles, have three types of plasma immunoglobulins. These three are small molecular weight two isoforms with high molecular weight immunoglobulin lgM (duck 800 kDa) and respective sedimentation coefficients 7.8S (duck 180 kDa) and 5.7S (duck 130 kDa). . (ER Unanue et al. J. Exp. Med. 121: 697-714 (1965); HM Grey, J. Immunol 98: 811-819 (1967); B. Zimmerman et al. Biochemistry 10: 482-448 (1971) Evian lgG is often referred to as lgY because it is present in egg yolk 5.7S lgY is composed of a short heavy chain and is constitutively and antigenically 7.8S lgY (Fig. 1R> 1) is similar to the F (ab ′) ₂ fragment, and this fact is expressed as lgY (same as 7.8S lgY) and lgY (ΔFc) (same as 5.7S lgY). (KE Magor et al., J. Immunol. 149: 2627-). 633 (1992 years)).

In studies of infected or experimentally immunized birds, duck antibodies do not sacrifice the binding activity corresponding to the antigen, and include biological and functional effects, including complement binding and Fc receptor binding. Proved to be numerically lacking. (GW Litman et al., Immunochemistry 10: 323 (1973); TE Toth et al., Avian Dis. 25: 17-28 (1981)). This is due to the apparent lack of the Fc-equivalent region of the lgY (ΔFc) antibody, which is a qualitative major component of the duck antibody response.

Therefore, lgY (ΔFc) antibodies, such as morphological and functional analogs of F (ab ′) ₂ fragments, have found promised methods for producing antibodies and have material requirements suitable for antibody activity. If recognized, it would be a great benefit in immunological use.

Evian egg yolk antibody has been reported to disclose clinically effective properties similar to research and mammalian antibodies (eg, US Pat. Nos. 5,340,923,5,585,098,5,601,823,5). 976, 519). Egg yolk that can be obtained from laying hens is cheaper, more convenient and safer to handle than over-immunized mammalian plasma. More importantly, egg yolk antibodies can withstand the rigorous testing of modern animal protection regulations (A. Polson et al., Immunol. Commun. 9: 475 (1980); and B. Gottstein et al. al.). These facts suggest the possibility of using egg yolk as a commercial source of antibodies. However, egg yolk lipid-rich substances such as fatty acids, cholesterol and lecithin make the separation of egg yolk antibodies cumbersome and difficult. Various efforts have been made in this regard. For example, bacterial culture medium, pectin (Japanese Patent Laid-Open No. 64-38098 issued on Feb. 8, 1989), dextran sulfate (JC Jensenius et al., J. Immunol. Methods 46:63 (1981). )), Natural rubber (H. Hatta et al., J. Food Science 53: 425 (1988)), and polyethylene glycol (PEG) (A. Polson et al., Immunol. Invest. 14: 323). 1985); precipitation agent can be dissolved in water, and the like owned U.S. also No. 4,550,019 to) the A.Polson mainly nonaqueous biomolecules lipid and egg yolk microspores (bio bio-molecules) Precipitate, thereby harvesting a water-soluble phase rich in egg yolk antibodies The A. Hassl et al developed a two-step chromatography, including hydrophobic interaction chromatography and gel filtration chromatography, to further separate egg yolk antibodies from PEG-purified fractions (A. Hassl and H. Aspock, J. Immunol. Methods 100: 225 (1988)).

Akita et al. Have described an improved method for lgY separation in which egg yolk is diluted with a large amount of water and the resulting supernatant is subjected to gel filtration chromatography and / or ion exchange chromatography to extract egg yolk antibodies from bird eggs. (EM Akita et al., J. Immunol. Methods. 160: 207 (1993); and EM Akita and S. Nakai, J. Food Sci. 57: 629 (1993)).

However, all these studies and patents focus on the separation of the entire population of egg yolk antibodies (including at least lgY and zlgY (ΔFc)) from Evian eggs rather than only purifying lgY (ΔFc) antibodies. Furthermore, the lgY (ΔFc) antibody is present only in birds belonging to the Order Angerformes family including ducks and geese. However, the conventional method described above does not mention an effective lgY (ΔFc) antibody purification proposal.

Therefore, for a fast, cost-effective and highly productive method that easily separates the desired lgY (ΔFc) antibody from the Evian antibody pool while simultaneously maintaining the activity of the lgY (ΔFc) antibody. There is a need. In effect, purified lgY (ΔFc) antibody may act as a new type of F (ab ′) ₂ antibody used in various immunodiagnostics and immunotherapy.

Summary of the Invention As noted above, extensive research has been conducted to meet the industrial requirements of egg yolk antibodies. Successful separation of egg yolk antibody from Evian egg yolk can be easily accomplished by non-aqueous dissolution-adsorption chromatography using an uncharged absorber and / or a salting-out method that differentiates another isoform of a simple egg yolk antibody. Was discovered by chance today. According to the method of the present invention, highly purified egg yolk antibody, more specifically highly purified lgY (ΔFc), can be easily harvested in an economically superior manner and can be immediately and widely used in a wide variety of immunological uses. Can do.

Therefore, the object of the present invention is to (1) obtain a water fraction of egg yolk from eggs of poultry hens, and (2) salt out the water fraction with the first non-denaturing salt of the first concentration, Obtain the majority of the first isoform of antibody from the water fraction of step (1) and (3) adjust the water fraction resulting from step (2) with the second non-denaturing salt of the second concentration. In the method of selectively separating antibody isoform from egg yolk, wherein the majority of the second isoform of the antibody is salted out from the water fraction obtained as a result of step (2). It is an object of the present invention to provide a method for selectively separating antibody isoforms from yolk, provided that the first non-denaturing salt and the second non-denaturing salt are the same.

Another object of the present invention is to provide a method for separating antibodies from egg yolk by using an absorptive substance to remove most of the lipids and casein substances normally present in egg yolk.

Thus, the present invention (a) removes non-aqueous biomolecules and egg yolk granules from the egg yolk of poultry hen eggs, thus obtaining a water miscible fraction with egg yolk antibody (b) ) water-miscible fractions, yolk antibodies capable of absorbing non-aqueous solubility - passes uncharged absorber (waterinsoluble non-charged absorbent) stationary phase comprising an effective amount, for use as a fraction (c) It is an object of the present invention to provide a method for selectively separating antibody isoforms from egg yolk, comprising collecting a solution flowing through a stationary phase and (d) recovering egg yolk antibody from the flowing solution of step (c). .

The present invention (a) removes non-aqueous biomolecules and egg yolk granules from the egg yolk of poultry hen eggs, thus obtaining a water miscible fraction with egg yolk antibody, (b) water-miscible fraction is present in normal egg yolk water-miscible lipid was capable of absorbing non-aqueous solubility - passes uncharged absorber (water insoluble non-charged absorbent) stationary phase comprising an effective amount, (c The present invention provides a method for selectively separating antibody isoforms from egg yolk, comprising the steps of: e) circulating a stationary phase to obtain an eluate , and (d) recovering the yolk antibody from the eluate of step (c). .

According to the method of the present invention, a large amount of egg yolk antibody selective isoform, more specifically, lgY (ΔFc) antibody can be used for various industrial drugs, economically and efficiently and time-saving. Can be earned.

It is still another object of the present invention to provide methods for use in clinical and research of the produced lgY (ΔFc) antibody. The present invention is cost-effective and easy to prepare, and according to the present invention, lgY (ΔFc) antibodies have the advantage of being inactive against the complement system and the rheumatoid factor of mammalian plasma and are reciprocal against mammalian lgG. The response is weak and is therefore particularly suitable for use with immunological analytes, including mammalian plasma, with minimal damage. It will be appreciated by those skilled in the art that lgY (ΔFc) antibodies can be present in a single clinical reagent, research, and other uses, or in a commercial set as an active ingredient.

Another distinct object of the present invention is to provide reagents as immunoanalytes of etiological agents, including lgY (ΔFc) antibodies obtained by the methods of the present invention.

Detailed Description of the Invention Egg plasma antibodies are abundant in the plasma and eggs of birds. However, as mentioned above, antibodies collected from eggs are also preferred economically. Egg laying hens transfer both lgY and lgY (ΔFc) isoforms from plasma to yolk. In principle, duck eggs contain about 1-4 milligrams of lgY / ml and about 3-12 milligrams of lgY (Fc) / ml each in the yolk so that the total antibody content of a single egg is 15 ˜80 milligrams of lgY and 45-240 milligrams of lgY (ΔFc) are expected. The large amount of yolk produced in any case far exceeds the amount of plasma that can be safely obtained from birds. In addition, egg yolk antibody extraction can be done on a large scale without significant investment. According to the present invention, a method for efficiently separating an antibody from egg yolk is provided, in which a method called “adsorption chromatography” or “differential salting-out” that can be used alone or in combination with other methods. Is an essential step in separation.

As used herein, “adsorption chromatography” is defined as a type of separation method that uses a stationary phase and selectively absorbs and condenses a suitable solute from the fluid phase. In one aspect of the present invention, the non-water soluble and unfilled absorber serves as an effective component in the stationary phase to stop lipid impurities that are normally miscible with water in egg yolk.

"Adsorption chromatography" according to the invention, preferably partially purified process is preceded by a non-aqueous bio-molecules and large microspores most at that partially purified process and desirably eliminate inappropriate protein most in egg yolk, To do. All conventional methods for effectively achieving such objectives can be used effectively in the present invention, typically using aqueous buffers or water to obtain an antibody-rich aqueous phase, and PEG, dextran. Using sulfate or natural rubber such as sodium alginate, carrageenan, xanthan gum, co-precipitate unsuitable materials.

In a preferred embodiment of the invention, the egg yolk is first separated from the egg white and then washed with distilled water to remove as much egg white as possible. The yolk membrane encapsulating the yolk is pierced and the separated yolk fraction is then diluted with an effective amount of aqueous buffer or water to form the yolk suspension. Preferably, the collected egg yolk is diluted quantitatively from 2 parts to roughly 40 parts with aqueous buffer or distilled water, more preferably from 5 parts to 30 parts quantitatively per egg yolk. Turn into. The pH value is a decisive factor in the stage of partial purification (EM Akita to S. Nakai, J. Food Sci, 57: 629 (1993)). For optimal recovery of egg yolk antibody, it is desirable that the pH be set within a range of about 5-7. The temperature of this step is preferably within the range of about 0 to 60 degrees. The egg yolk suspension is gently agitated to make a similar mixture and then left for a period of time sufficient to make the aqueous and non-aqueous phases. Non-aqueous dissolved material, including lipoproteins, phospholipids, sterols and equivalent non-aqueous biomolecules, is then removed from the aqueous egg yolk suspension by centrifugation. The resulting antibody containing supernatant is then separated from the cohesive precipitant by decanting, absorption, or other methods well known to those skilled in the art. In general, the lipid content of the water-soluble fraction thus obtained is still so high that it is disadvantageous for the subsequent operation. According to the present invention, the fluctuating phase comprising the non-water soluble uncharged absorber is cultured with a sufficient amount of water soluble fraction to adsorb the water soluble lipid material remaining on most water soluble materials. Suitable absorbers include, but are not limited to, fume silica, amorphous silicic acid, silica dioxide, silica gel, silicate, diatomaceous earth, acid clay, talc, alumina, activated carbon, aluminum oxide, titanium oxide, And other material-lipid-absorbable compounds or natural rough wall soil. Particularly preferred absorbers are fume silica, silicic acid dioxide, diatomaceous earth. The rate of absorption into the water-soluble fraction varies widely depending on the nature of the absorber selected. When fume silica is used in the process, it is desirable to increase the weight by 0.1% or more, and more preferably, increase the weight by 0.3-5% based on the amount of water soluble fraction to be treated. It is desirable. When the absorbent is silicic acid dioxide or diatomaceous earth, the absorption chromatography of the present invention is preferably carried out in the range of 3% to 20% based on the weight of 1% or more, more preferably the amount of the water-soluble fraction to be treated. The absorption chromatography according to the present invention is a lipid substance retained on the surface of the absorber, such as batch treatment of the water-soluble fraction using the absorber, or flowing the water-soluble fraction over a chromatography column packed with the absorber. Any conventional method can be used as long as the amount is sufficient. The reaction time and reaction temperature during the treatment do not have a fatal effect on the result, and a reaction temperature of 4 to 30 degrees and a reaction time of 10 to 60 minutes are usually feasible. The absorption method can be repeated several times with each new absorber, if necessary, but usually only one operation is sufficient. In this way, lipids and most non-lipid substances can be successfully separated into two immiscible phases.

The ability of the selected absorbent to obtain immunoglobulins, the yolk antibodies can be recovered from the "Flow Through" represented by means of the solution passing through the stationary phase effluent or here eluted from the stationary phase. As shown in the preferred embodiment herein, egg yolk antibody is more than 90% diatomaceous earth when fume silica or silicic acid dioxide is used as an absorber, whereas egg yolk antibody is usually present in the stationary phase. The antibody is left in the fluid. The selection of a special method for eluting egg yolk antibodies is determined by one skilled in the art. Generally, the eluate pH is buffered with a value greater than 4, or less than 8, or eluate containing chaotropic reagents, without sufficiently dissociate lipid material from the stationary phase, for eluting the antibody from the stationary phase In the present invention, an eluate containing an antibody is formed. As used herein, the term “ eluate ” refers to a liquid that contains the desired substance dissolved by the eluate from the stationary phase. The terms “chaotropic reagent” or “chaotrope” are drugs that can induce conformational changes in protein molecules such as antibody molecules and are therefore well known as protein denaturing agents. According to the present invention, the majority of bound antibody can be successfully eluted using any neutral buffer containing moderately enriched (1> M) chaotropic reagents. In many applications, removing the chaotrope after elution will restore the natural protein composition.

Useful eluents include, but are not limited to, glycine-HCl 0.1 M, pH 2.3; glycine-HCl 0.1 M, pH 10.0; guanine-HCl 6 M, pH 3.0; potassium chloride. 3.0M; potassium iodide 5M; magnesium chloride 3.5M; ammonium / sodium / potassium thiocyanate 1-3M, urea 6M and the like. However, with regard to the activity of the collected antibody, it has a moderate ionic strength, such as sodium thiocyanate 3M buffered with 20 mM MES buffer (pH 5.8) or 20 mM tris (hydroxymethyl) -aminomethyl, and has a chaotrope. Including neutral pH buffer is more suitable for practicing the present invention. The activity state of the collected antibody can be easily recovered, for example, by dialysis extensively against low ionic strength, no chaotrope, or weak acidic buffer. As one feature of the present invention, the antibody-concentrated eluate or fluid can then be subjected to a differential salting-out step to separate the egg yolk antibody isoform. In this text, the term “salting out” has a common meaning in the field of protein science, and refers to an invariant salt that is added to a mixture or production batch to reduce the solubility of the protein. Lead to condensation. The term “differential salting out” refers to a salting out method in which two or more proteins are differentially precipitated or coagulated from a mixture by changing the salting or salt concentration. In the present invention, proteins that undergo differential salting-out are egg yolk antibody isoforms, such as lgY and lgY (ΔFc). Examples of invariant salts useful for the precipitation of egg yolk antibodies are, but not limited to, NaCl, Na ₂ SO ₄ , (NH 4) ₂ SO ₄ , KCl, CaCl ₂ , and MgSO ₄ . Preferably, the invariant salt is most preferably Na ₂ SO ₄ or (NH ₄ ) ₂ SO ₄ , and (NH ₄ ) SO ₄ . The salinity of the differential precipitation of egg yolk antibody isoform depends on the type of salt and can be determined through simple tests performed by one skilled in the art. If (NH ₄ ) ₂ SO ₄ is used in the preferred embodiment of the present invention, lgY is salted out at a salt concentration of 21% (w / v) or less of the treated eluate or fluid, and lgY ( ΔFc) rises to a salt concentration of about 31% (w / v) of the treated eluate or fluid. It can also be appreciated that the order of precipitation of the two antibody isoforms also varies with the choice of salt. In this operation, it is also possible to precipitate a first isoform with a combination of two or more salts, for example, one salt first and then the second isoform with another salt. The differential precipitation method of the present invention greatly accomplishes the main objective of the present invention, eg, the essential separation of the desired lgY (ΔFc) antibody from the entire population of egg yolk antibodies composed of both lgY and lgY (ΔFc). Will be. If higher purity antibody acquisition is desired, the precipitated antibody can be re-dissolved in a suitable buffer system, eg additional gel filtration chromatography, hydrophobic interaction chromatography, ion exchange chromatography, immunoaffinity chromatography, etc. What is necessary is just to perform a purification | cleaning means. As used herein, the term “immunoaffinity purification method” or “immunoaffinity chromatography” refers to a separation method based on the binding properties of an antibody for a particular antigen. That is, an antibody that binds to a specific antigen under a certain condition is separated from an antibody that has melted under that condition. The present invention is intended for use in immunoaffinity purification to eliminate irrelevant proteins, and in particular removes non-antigen binding immunoglobulins.

According to the present invention, immunoaffinity purification is carried out using an “antigen matrix” consisting of an antigen immobilized on an insoluble support. The type of support is not critical in the immunoaffinity purification of the present invention. Conventional support materials suitable for covalent attachment of antigens are useful for interventions in the interaction of the desired antibody with the antigen immobilized thereon. Usually, the support consists of cross-linked agarose or cross-linked dextran, such as CNBr-activated Sepharose 4B, available from Pharmacia. The antibody purified by differential salting-out is dissolved in a “binding buffer” and applied onto the antigen matrix so that an immunocomplex of fixed antigen and an egg yolk antibody are formed. Both buffer systems intercalated with antigen-antibody interactions and buffer systems that are effective in maintaining the desired binding state are useful in the present invention. Preferably, the binding buffer is selected from the group consisting of phosphate buffer, MES (2- [N-morpholine] ethanesulfonic acid) buffer, and Biz-Tris buffer, with 20 mM MES buffer being the most. Is preferred.

The immunoaffinity purification is preferably performed in an environment with a weak acid and low ionic strength. For example, an environment in which the pH range is within 4 to 7 and the ionic strength is lower than 50 mM. More preferably, the antibody allows interaction with the immobilized antigen in a pH range of 5-6, optimally in the range of 5.6-5.8. Egg yolk antibodies can be dissociated from the antigenic matrix by chaotropic salts or pH below 4 or above 8. The activity of the collected antibody can be restored by, for example, large-dialysis against a weak acid buffer with low ionic strength and no chaotrope.

The lgY (ΔFc) purification in the present invention does not activate the complement system and does not lead to a factor in mammalian plasma rheumatism. The immunological interaction between lgY (ΔFc) and mammalian lgG is not critical. Therefore, the present invention provides a new type of antibody suitable for clinical use and research use.

The present invention also provides a wide variety of clinical and research uses of lgY (ΔFc) antibodies prepared in accordance with the present invention. For example, the present invention provides a method of immunizing an animal or patient (including poultry, livestock, and companion animals) with a therapeutic dose of an lgY (ΔFc) antibody of the present invention. The present invention relates to various pathogenic agents including microorganisms such as bacterial, natural, recombinant or peptide synthetic pathogens, fungi, protozoa, nematodes, etc., and natural, recombinant or peptide synthetic allergens, It protects against proteins or non-protein substances such as toxins, venoms, hormones, or other immunogens that can elicit an immune response. Preferably, the purified lgY (ΔFc) antibody is provided with a pharmaceutically acceptable carrier such as water, salt, etc.

The lgY (ΔFc) antibody of the present invention is also useful for detection of important pathogens, for example, pathogenic or non-pathogenic organisms such as Escherichia coli and Gertonel, and other bacterial organisms; natural, recombinant Type or peptide synthetic hormones such as estrogen, progesterone, radiothyroxine; major histocompatibility complex antigens, etc .; natural, recombinant, or alpha-fetoprotein, prostate antigen and such peptide synthetic tumor markers; C -Disease state markers such as reactive protein, ferritin, etc .; examples in the body such as fluids, tissues, cell extracts, etc., derived from humans and animals.

Using lgY (ΔFc) antibodies obtained according to the present invention, the etiological agent can be detected quantitatively or qualitatively using methods well known to those skilled in the art. For example, octaroni method (MO), one-way radial immunodiffusion method (SRID), immunoelectrophoresis method (IEP), radioimmunoassay method (RIA), ELISA method (ELISA), Western blot method (WB), turbidimetric analysis Immunoassay, enzyme immunoassay, turbidimetric immunoassay, chemiluminescence assay immunoassay, immunogold assay, or immunochromatography assay.

The lgY (ΔFc) antibody of the present invention can also be adapted for use in biochips and biosensors.

Preferred Embodiments of the Invention The following examples are for the purpose of illustrating the invention and are not disclosed for the purpose of limiting the scope of the invention.

Example 1: Immunization Method for Stimulation of Specific Antibody Production Twelve weekly domestic ducks (Anas platyrhynchosvar. Domestica) 16 weeks old were housed for antibody and egg production, respectively. First of all, the duck was human C-reactive protein (CRP; purified from human ascites) 1-5 mg / ml in phosphate buffer, emulsified at the pH 7.5 with the same amount of Freund's complete adjuvant. It is injected subcutaneously. The concentration of antigen used is usually in the range of 1-5 mg / ml. After the first injection, young hens receive three additional injections of 1-5 mg / ml of antibody every two weeks. One week later, eggs begin to be collected, labeled, and stored at 4 degrees Celsius until subjected to antibody extraction and purification operations. The booster method (booster method) is repeated every four weeks during the experiment. Seven days after each booster injection, blood is collected. Each blood sample is centrifuged and the resulting plasma is collected.

Example 2: Antibody extraction from duck egg yolk Egg yolk collected from the egg laid by the hyperimmune duck of Example 1 is washed thoroughly with a weak jet of distilled water to remove protein glands. After measuring the amount of egg yolk, mix thoroughly with distilled water 10 times the amount of egg yolk measured. The mixture is kept at 4 degrees Celsius for a minimum of 2 hours and then centrifuged in a Hitachi CR-22F centrifuge at a speed of 10,000 rpm for 1 hour. A pale supernatant and a semi-solid flexible layer are formed in the centrifuge tube.

Example 3: Treatment with Absorber One of the absorbers is added to the crude extract prepared in Example 1: 2% (w / v) fume silica (purchased from Sigma), 3% (w / v) Silica dioxide (Sigma), 3% (w / v) celite diatomine (purchased from Reset), or 5% (w / v) celite diatomin hyflocel (Cerit). The resulting suspension is cultured with gentle agitation at 4 degrees Celsius for 60 minutes. The absorbent after completion of the culture is precipitated at a speed of 20,000 rpm at 4 degrees Celsius using a Hitachi CR-22F centrifuge, and the supernatant and pellet are harvested separately. Each 10 μl sample taken from the supernatant is subjected to non-reduced sodium dodecyl sulfide polyacrylamide gel electrophoresis (SDS-PAGE).

As shown in FIG. 1, with respect to the amount of antibody absorbed by the absorber, fume silica performs the maximum absorption activity, and the antibody left in the fluid is nearly nothing. Silica dioxide appears to be due to its low porosity (because of its low gross surface area), but it has a slightly weaker affinity for immunoglobulins than fume silica. Conversely, with either type of diatomaceous earth, less than 10% of the antibody was obtained.

Example 4: Differential Salting-out of Egg Yolk Antibody The fume silica pellet in Example 3 is treated with 2.5 M sodium tisocyanate (pH 7.5) to elute the antibody bound thereon. The resulting eluate is first precipitated with ammonium sulfate at a concentration of 21% (w / v) of the eluate volume and then second precipitated with an additional 31% (w / v) concentration of ammonium sulfate. Is done. The precipitated antibody is redissolved in phosphate buffer (PBS). An SDS-PAG analysis of 8% non-reducing acrylamide gel is performed. This includes 2237 μg crude extract from Example 2 (lane 1), 10 μl harvested fluid from example 3 (lane 2), 1122.25 μg eluate from the fume silica pellet from (lane 3), And 153 μg and 372.85 μg of antibody product (lane 4 and lane 5 respectively) harvested from the first and second precipitation steps are loaded. The results are shown in Table 2. The percent regeneration and cleanup was measured by scanning the gel concentration, which is summarized in Table 1.

As shown in Table 1, the resulting lgY (ΔFc) antibody has a clearance value greater than 96% and is regenerated with a yield of approximately 76% (72.05 mg / 119.8 mg × 100%) . More importantly, this purification strategy leads to essentially separating the desired lgY (ΔFc) antibody from the whole egg yolk antibody composed primarily of both lgY and lgY (ΔFc).

Example 5: Utilizing the ability of diatomaceous earth to attract the immunoaffinity purified lipid of egg yolk antibody and reject the antibody, the crude extract prepared in Example 2 is a 10% weight sheet based on the amount of crude extract injected. It is injected onto the filtration column with light atomin. The solution flowing through the column is collected and first precipitated with 21% (w / v) ammonium sulfate of the amount of fluid in the solution. The precipitated antibody is collected and the supernatant is bisected. A portion of the supernatant is precipitated with about 31% concentration of ammonium sulfate, and the other portion of the supernatant is precipitated with 16 (w / v) sodium sulfate. The precipitated antibody is redissolved in PBS. SDS-PAGE analysis was performed on an 8% non-reducing aclereamide gel, collected from 2012.5 μg of crude extract from Example 2 (lane 1), filtration of 1678 μg of celite diatomine (lane 2). Antibody collected from the second precipitation step according to the flow, 94.9 μg of the first precipitation step (lane 3) and 31% ammonium sulfate and 16% sodium sulfate (lane 4-5) Things are loaded. The result is shown in FIG. The percent recovery and cleanup was determined by scanning and measuring the gel concentration and is summarized in Table 2.

As shown in Table 2, the resulting lgY (ΔFc) antibody is approximately 77% (when sodium sulfate is used in the second precipitation step), and ammonium sulfate is used in the second precipitation step. Both are recovered at a high rate of about 69%.

Example 6: Immunoaffinity-purified C-reaction protein (CRP) solution of egg yolk antibody is prepared in 0.1 M carbonate buffer at a concentration of 5 mg / ml and pH 8.5. CNBr reaction Sepharose 4B purchased from Pharmacia is first washed with 10 mM cooled HCI 1 mM in matrix and allowed to react overnight at 4 degrees Celsius with CRP solution in matrix twice. The antigen matrix is retained at 4 degrees Celsius for 2 hours in 0.5 M ethanolamine solution in 20 mM Tris-HCl (pH 8.5) with a one-to-one proportional value. The antigen matrix is then washed with PBS containing 0.02% sodium azide and stored at 4 degrees Celsius.

In Example 4 a duck antibody and antigen matrix precipitated with 21% (w / v) ammonium sulfate is used. 1 ml of the antigen matrix is packed in a conventional column and invaded by 20 mM MES (2- [N-morpholino] ethanesulfonic acid) buffer (pH 5.8). The antigen matrix allows reaction with 0.25 ml of antibody formulated in the same binding buffer. The antigen matrix is washed with binding buffer until there is almost no protein in the eluate. The bound antibody is immediately eluted with 6M guanidine-HCl, so its optical density is measured at 280 nm after complete dialysis. Analytical SDS-PAGE is shown in FIG. 4 and shows that the affinity-purified antibody is composed primarily of lgY (ΔFc) antibodies represented by a single band on the gel.

All patents and references cited herein are hereby incorporated by reference herein in their entirety. In the event of a conflict with a cited reference, the matters described herein, including the definition, prevail. While the invention has been described with reference to the specific embodiments described above, it will be appreciated that various modifications and changes obvious to those skilled in the art can be made without departing from the spirit and scope of the invention. Should.

The above and other objects and features of the present invention will become apparent by referring to the following preferred embodiments and the accompanying drawings.

The SDS-PAGE analysis shows an absorber with four antibody acquisition capacities: where lane 1 is a partially purified antibody extract; lane 2 is 2% FUMED SILICA Lane 3 is liquid flowing 3% silica dioxide; lane 4 is liquid flowing 3% CELITE DIATOMITE; lane 5 is 3% seawater Liquid in circulation through CELITE DIATOMITE FYFLO-CEL; Lane 6 is liquid in circulation through 5% sealite diatomite hyflocell; Result of electrophoresis of purified egg yolk antibody passed through 8% SDS polyacrylamide gel (POLYACRYLAMIDE GEL) with fume silica as an absorber: where lane 1 is a partially purified antibody extract; lane 2 is the liquid flowing through 2% fume silica; lane 3 is the eluate from the fume silica pellet; lane 4 is precipitated with 21% (w / v) ammonium sulfate in the first precipitation step Lane 5 shows the antibody product precipitated with 31% (w / v) ammonium sulfate in the second precipitation step; Electrophoresis result of purified egg yolk antibody passed through 8% SDS polyacrylamide gel with celite diatomine as an absorber; Lane 1 is partially purified antibody extract; Lane 2 is Light diatomine filtrate; Lane 3 is antibody product precipitated with 21% (w / v) ammonium sulfate in the first precipitation step; Lane 4 is 31% (w / v) ammonium sulfate in the second precipitation step Lane 5 shows the antibody product precipitated with 16% (w / v) sodium sulfate in the second precipitation step; and Electrophoresis result of purified egg yolk antibody passed through 8% SDS polyacrylamide gel with celite diatomine as absorber; M is molecular weight marker; lane 1 is partially purified egg yolk antibody Extract; Lane 2 is 2% fume silica circulating liquid; Lane 3 is eluate from fume silica pellets; Lane 4 is 21% (w / v) ammonium sulfate precipitated antibody production Lane 5 is the antibody product purified by bioaffinity chromatography. It is a figure which shows the result of the absorptive power of nine types of non-water-decomposability-unfilled absorbers. It is a figure which shows the result of the absorptive power of nine types of non-water-decomposability-unfilled absorbers.

Claims (35)

(A) removing non-aqueous biomolecules and egg yolk granules from the egg yolk of poultry hen eggs, thereby obtaining a water miscible fraction with egg yolk antibody;
(B) water-miscible fractions, yolk antibodies capable of absorbing non-aqueous solutions of - passing the non-charged absorbent (water insoluble non-charged absorbent) stationary phase comprising an effective amount,
(C) collecting the solution that has flowed through the stationary phase for use as a fraction;
(D) including the step of recovering the yolk antibody from the circulating solution in the step (c),
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 1,
The removal step (a) dilutes the yolk with an effective amount of a dilute agent selected from the group consisting of an aqueous buffer and water,
Then, by separating the resulting diluted egg yolk,
Comprising removing non-aqueous biomolecules and egg yolk granules ,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 2,
The effective amount of the diluted agent is in the range of 5 to 30 parts per part of egg yolk,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 1,
The absorber is characterized by being diatomaceous earth,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 1,
(1) obtaining a majority of the first isoform of the antibody from the flowing solution of step (c) by salting out the flowing solution with the first undenatured salt of the first concentration;
(2) When most of the second isoform of the antibody is salted out from the product obtained as a result of step (1) with the second non-denaturing salt of the second concentration, the first non-denaturing salt and the second non-denaturing salt. If the salt is the same, the first and second concentrations are different.
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 5,
The first unmodified salt and the second unmodified salt are each selected from a group consisting of NaCl, Na ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ , KCl, CaCl ₂ and MgSO _4. Characterized by the
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 5,
If the first unmodified salt and the second unmodified salt are the same,
The second concentration is higher than the first concentration,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 5,
When (NH ₄ ) ₂ SO ₄ is used as the only unmodified salt,
Based on the amount of water fraction treated in step (1) and the amount of water fraction treated in step (2),
The first concentration is 21 % (w / v) or less,
A method for selectively separating antibody isoforms from egg yolk, wherein the second concentration is 31 % (w / v).
A method for selectively separating antibody isoforms from egg yolk according to claim 7,
When (NH ₄ ) ₂ SO ₄ is used as the first unmodified salt,
Based on the amount of water fraction treated in the step (1), the first concentration is 21% (w / v) or less,
Based on the amount of water fraction that the second concentration was treated in step (2),
Characterized in that it is achieved by adding 16 % (w / v) Na ₂ SO ₄ as the second unmodified salt,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 5,
Wherein the first isoform of the antibody is lgY and the second isoform is lgY (ΔFc),
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 1,
An antibody selective to egg yolk characterized by being followed by one or more purification methods selected from the group consisting of gel filtration chromatography, hydrophobic interaction chromatography, ion exchange chromatography, and immuno-affinity chromatography How to separate isoforms.
A method for selectively separating antibody isoforms from egg yolk according to claim 11,
At least one purification method is immuno-affinity chromatography,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 12,
Immuno-affinity chromatography is carried out at pH 4-7 and ionic strength lower than 50 mM,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 13,
Immuno-affinity chromatography is carried out at a pH of 5.6 to 5.8,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 1,
Egg yolk is obtained from eggs of poultry hens immunized with selected antigens,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 15,
The poultry hen is a duck,
A method of selectively separating antibody isoforms from egg yolk.
(A) removing non-aqueous biomolecules and egg yolk granules from the egg yolk of poultry hen eggs, thereby obtaining a water miscible fraction with egg yolk antibody;
(B) water-miscible fractions, yolk antibodies capable of absorbing non-aqueous solutions of - passing the non-charged absorbent (water insoluble non-charged absorbent) stationary phase comprising an effective amount,
(C) flow through the stationary phase to obtain the eluate ,
(D) collecting the yolk antibody from the eluate of step (c),
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 17,
The removal step (a) dilutes the yolk with an effective amount of a dilute agent selected from the group consisting of an aqueous buffer and water,
Then, by separating the resulting diluted egg yolk,
Comprising removing non-aqueous biomolecules and egg yolk granules ,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 18,
The effective amount of the diluted agent is in the range of 5 to 30 parts per part of egg yolk,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 17,
The absorber is selected from the group comprising fumed silica and silica dioxide,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 20,
The egg yolk antibody in step (c) is characterized in that the chaotropic salt or eluate is eluted from the stationary phase at a pH value of 5.8 ,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 21,
The chaotropic salt is selected from the group comprising 4M guanidine HCl and 1-3M thiocyanate,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 17,
(3) obtaining a majority of the first isoform of the antibody from the eluate of step (c) by salting out the water fraction with the first undenatured salt of the first concentration;
(4) When most of the second isoform of the antibody is salted out from the product obtained as a result of step (1) with the second non-denaturing salt of the second concentration, the first non-denaturing salt and the second non-denaturing salt. When the salt is the same, the step (c) includes a sub-step on the condition that the first concentration and the second concentration are different.
A method of selectively separating antibody isoforms from egg yolk.
The method of selectively separating antibody isoforms from egg yolk according to claim 23,
The first unmodified salt and the second unmodified salt are each selected from a group consisting of NaCl, Na ₂ SO ₄ , (NH ₄ ) ₂ SO ₄ , KCl, CaCl ₂ and MgSO _4. Characterized by
A method of selectively separating antibody isoforms from egg yolk.
The method of selectively separating antibody isoforms from egg yolk according to claim 23,
If the first unmodified salt and the second unmodified salt are the same,
The second concentration is higher than the first concentration,
A method of selectively separating antibody isoforms from egg yolk.
The method of selectively separating antibody isoforms from egg yolk according to claim 23,
When (NH ₄ ) ₂ SO ₄ is used as the only unmodified salt,
Based on the amount of eluate processed in step (1) and the amount of product obtained as a result of processing in step (2), the first concentration is 21 % (w / v) or less,
A method for selectively separating antibody isoforms from egg yolk, wherein the second concentration is 31 % (w / v).
The method of selectively separating antibody isoforms from egg yolk according to claim 23,
When (NH ₄ ) ₂ SO ₄ is used as the first unmodified salt,
Based on the amount of eluate processed in the step (1), the first concentration is 21% (w / v) or less,
Based on the amount of product obtained as a result of the second concentration being processed in step (2) above,
Characterized in that it can be achieved by adding 16 % (w / v) Na ₂ SO ₄ as the second unmodified salt,
A method of selectively separating antibody isoforms from egg yolk.
The method of selectively separating antibody isoforms from egg yolk according to claim 23,
The first isoform of the antibody is lgY;
Wherein the second isoform is lgY (ΔFc),
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 17,
An antibody selective to egg yolk characterized by being followed by one or more purification methods selected from the group consisting of gel filtration chromatography, hydrophobic interaction chromatography, ion exchange chromatography and immuno-affinity chromatography How to separate isoforms.
A method for selectively separating antibody isoforms from egg yolk according to claim 29,
At least one purification method is immuno-affinity chromatography,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 30,
Immuno-affinity chromatography is carried out at pH 4-7 and ionic strength lower than 50 mM,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 31,
Immuno-affinity chromatography is carried out at a pH of 5.6 to 5.8,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 17,
Egg yolk is obtained from eggs of poultry hens immunized with selected antigens,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to claim 33,
The poultry hen is a duck,
A method of selectively separating antibody isoforms from egg yolk.
A method for selectively separating antibody isoforms from egg yolk according to any one of claims 1 to 34,
The prepared antibody lgY (ΔFc) is not an Fc equivalent region,
A method of selectively separating antibody isoforms from egg yolk.