CN1463985A - Process for selectively separating IgY antibody from anseriforme birds eggs and IgY antibody produced therefrom - Google Patents
Process for selectively separating IgY antibody from anseriforme birds eggs and IgY antibody produced therefrom Download PDFInfo
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- CN1463985A CN1463985A CN 02123237 CN02123237A CN1463985A CN 1463985 A CN1463985 A CN 1463985A CN 02123237 CN02123237 CN 02123237 CN 02123237 A CN02123237 A CN 02123237A CN 1463985 A CN1463985 A CN 1463985A
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Abstract
The present invention is the method of separating and purifying yolk antibody from egg of anseriformes bird. The required yolk antibody is separated through adsorption and chromatographic step with one kind of water unsoluble and un-charged adsorbent, and the IgY antibody is deposited via salting-out step. The present invention also relates to the yolk antibody and its various application.
Description
Technical field
The present invention relates generally to a kind of method from Anseriformes birds yolk sharp separation and purifying yolk antibody (particularly IgY (Δ Fc) antibody), and the yolk antibody that method obtained by this.In detail, the present invention relates to a kind of from Anseriformes birds yolk, the separation and the method for purifying yolk antibody, it is by utilizing a water insoluble and non-charged sorbent material to carry out the adsorption chromatography flow process, the separation of the yolk antibody of being desired to reach, and be settled out these IgY by the flow process of saltouing with coming distinctiveness.The present invention also relates to the purposes of these IgY antibody on quantitative or qualitative immunoassay, or is directed to a cause of disease antigen prepd medical composition interested.
Background technology
Antibody has been widely used in multiple biological study and clinical application.The serum that is obtained by the hyperimmunization Mammals is the modal source of polyclonal antibody.Wait the antibody of immune serum gained to belong in the proteinic group that generally is called ＂ immunoglobulin (Ig) ＂ thus, in this immunoglobulin (Ig), immunoglobulin G (IgG) is maximum.The IgG molecule is made up of three zones (domains), and two Fab zones and a Fc zone are wherein arranged.The Fab position relates generally to antigenic combination.Though the Fc position does not have and antigen bonded ability, it is the multiple biological activity of management antibody, such as the combination of complement in conjunction with (complement fixing) and Fc acceptor.
In the technology of immunodiagnostics, one complete IgG molecule also is not suitable among the detection system and immunoassay relevant with mammalian blood serum, its be because the Fc zone on the IgG molecule have with the Fc receptors bind, activate this complement system and with abilities such as Rheumatoid factors, polyclonal (rheumatoid factor) in the mammalian blood serum reacts.General normal this kind interferential that causes that removes at the Fc position of IgG molecule reduces people such as (be published in ＂ Enzymology method (Methods in Enzymology) ＂, the 121st volume, 652-663 page or leaf in 1986) E.Lamoyi.
In immunotherapy, some suggestion of antibody is used and to be comprised that treatment suffers from the sufferer of the bacteriotoxin or the snake venom of poisoning property (as United States Patent (USP) case number the 5th, 340,923 and 5,601, in No. 823), and the piggy of birth just to the prevention of fatal enteron aisle colibacillosis (people such as H.Brussow, J.Clin.Microbiol. the 25th rolls up, 982 pages (1987); And people such as C.O.Tacket, NewEng.J.Med. the 318th volume, 1240 pages (1988)).The Fc section of a known antibody molecule is position people such as (, J.Immunol.Methods. the 162nd volume, 155-164 page or leaf (1993)) E.M.Akita of the tool antigenicity of this immunoglobulin (Ig), and (it can cause F (ab ') in the then excision of this section
2The formation of section) will obviously reduce majority and be positioned at potential anaphylactogen position on the immunoglobulin molecules, be useful to the mankind or the animal of using this immunoglobulin (Ig) therefore.
The F (ab ') that has shown this pair bond recently
2The antibody section is applicable in the immunodiagnosis test (people such as M.Muratsugu, J.Colloid Interface Sci the 147th volume, 378 pages (1991); And people such as J.L.Ortega-Vinuesa, J.Immunol Methods the 90th volume, the 29th page (1996)), and more be applicable to than IgG itself in the development of the immunoassay relevant with mammalian blood serum.
Yet, F (ab ')
2The antibody section is not as desired being widely used in the clinical immunodiagnosis cover group of common people.It may be owing to this F (ab ')
2The degree of difficulty of the scale operation of section and the reason that does not meet economical efficiency, this F (ab ')
2Section be traditionally by IgG through pepsic hydrolysis, and after purification by chromatography and making.
Close person of taxonomy co-relation such as duck and its and part reptilia (such as tortoise) have three kinds of serum immune globulins: i.e. giant molecule Immunoglobulin IgM (being 800kDa in the duck), and each two kinds of settling ratio is 7.8S (being 180kDa in the duck) and the isomeric form of the lower molecular weight IgG of 5.7S (being 130kDa in the duck) (people such as E.R.Unanue, J.Exp.Med. the 121st volume, 697-714 page or leaf (1965); H.M.Grey, J.Immunol the 98th volume, 811-819 page or leaf (1967); And people such as B.Zimmerman., biological chemistry (Biochemistry) the 10th volume, 482-448 page or leaf (1971)).Because birds IgG is present in the yolk, be commonly referred to as IgY.By the IgY of the 5.7S that heavy chain constituted of weak point F (ab ') on structure and antigenicity similar in appearance to 7.8S IgY
2Section (as shown in Figure 1), the isomeric form that this fact causes utilizing IgY (being equivalent to 7.8S IgY) and IgY (Δ Fc) name of (being equivalent to 5.7SIgY) to represent two kinds of IgY people such as (, J.Immunol. the 149th volume, 2627-2633 page or leaf (1992)) K.E.Magor.
Infect or experimental immunization birds in carried out studies show that the most biological effect functions of this duck antibody deficiency, comprise the combination of complement and the combination of Fc acceptor, but it is antigenic in conjunction with active (people such as G.W.Litman, Immunochemistry the 10th volume, 323 pages (1973) for correspondence not sacrifice its grade; And people such as T.E. Toth, Avian Dis. the 25th volume, 17-28 page or leaf (1981)).Its obviously lacking of Fc-equity zone that may be due to this IgY (Δ Fc) antibody, do not have, and this IgY (Δ Fc) antibody constitutes the major portion of this Anseriformes avian antibodies reaction.Thereby believe, if can find the method for trustworthy this antibody of manufacturing, and can identify the physical property important document that is suitable for its activity, then be somebody's turn to do and F (ab ')
2Section can provide benefit out of the ordinary at IgY (Δ Fc) antibody similar on structure and the function on immunity is used.
Through report, the birds yolk antibody has useful characteristic in research and the clinical application as Mammals antibody.(as U.S. Patent number the 5th, 340, No. 923, U.S.5,585, No. 098, U.S.5,601, No. 823 and U.S.5,976, No. 519).By the yolk of the hen gained of laying eggs than the hyperimmunization mammalian blood serum more cheap and operation go up more convenient and safer.The more important thing is that yolk antibody can conform to relevant law standard (people such as A.Poison, Immunol.Commun. the 9th volume, 475 pages (1980) of modern protection of animal; And people such as B.Gottstein).The potential use of these true hint yolk is as the commercial source of antibody.
Yet, high-load in the yolk such as lipid such as lipid acid, cholesterol and Yelkin TTS, make and separate the work that yolk antibody becomes trouble and difficulty.Many effort have been devoted to this.For example, the aqueous precipitation thing, comprise agar, mucilage (in disclosed day on February 8th, 1989 in disclosure case 64-38098 number), T 500 (people such as J.C.Jensenius, J.Immunol.Methods the 46th volume, 63 pages (1981)), natural gum (people such as H.Hatta, Food science magazine (J.Food Science) the 53rd volume, 425 pages (1988)) and polyoxyethylene glycol (PEG) (people such as A.Polson, Immunol.Invest. the 14th volume, 323 pages (1985); Also be found in No. the 4th, 550,019, the United States Patent (USP) giving A.Polson) etc. material be used to precipitate non-aqueous biomolecules, be mainly lipid and yolk particle, contain the water soluble phase of enriching the yellow antibody of whole egg group to collect one by this.People such as A.Hassl have been developed a kind of two step layer analysis methods, it comprises hydrophobic interaction chromatography method and size exclusion chromatography method, further in the part isolate of PEG-purifying, to isolate this yolk antibody (A.Hassl and H Aspock certainly, JImmunol, Methods. the 110th volume, 225 pages (1988)).People such as Akita have described a kind of method that is used for the improvement of separating IgY, wherein the yolk antibody from ovum gallinaceum dilutes this yolk by the water with a large amount of volumes, then the supernatant liquor that is obtained is carried out size exclusion chromatography method and/or ion exchange chromatography, and in egg, extract and get (people such as E.M.Akita, J.Immunol.Methods. the 160th rolls up, 207 pages (1993); And E.M.Akita and S.Nakai, J.Food Sci. the 57th volume, 629 pages (1993)).
Yet all research and patent all concentrate on isolates all yolk antibodies (it comprises the IgY that has at least and do not have the Fc zone) from birds yolk, go out IgY (Δ Fc) and IgY antibody but not be absorbed in selective separation.Moreover, because of IgY (Δ Fc) antibody exists only in Anseriformes (Order Anseriformes) birds such as comprising duck and goose, and because the fat content in the Anseriformes birds yolk is higher than according to reports such as galliformes birds such as chicken and turkey (galliform birds), so the aforementioned conventional method also can't provide any hint for the successful purifying of IgY (Δ Fc) antibody.IgY (Δ Fc) antibody only once went out with the IgY co-precipitation in duck serum, and this method was both complicated and expensive, but still did not have independent IgY (Δ Fc) antibody and isolated by selectivity in yolk.
Therefore, have a demand for the method for quick, low cost and high benefit in the prior art, the IgY antibody that this method can simple and easy separation be desired in all antibody of Anseriformes bird egg can be kept the activity of this IgY antibody simultaneously.The IgY of this essence purifying (Δ Fc) antibody can be used as a kind of F (ab ') of novel type
2Antibody is for the usefulness of various immunodiagnosises and immunotherapy.
Summary of the invention
The inventor has carried out extensive studies, to satisfy above-mentioned industry demand to yolk antibody.Now, finding successfully to isolate from birds yolk yolk antibody unexpectedly can be reached easily, it is via the adsorption chromatography flow process of using a water insoluble and non-charged sorbent material, and/or via the flow process of simply saltouing that can distinguish the different isomerization thing of yolk antibody.According to the inventive method, can under the economic benefit of high yield, obtain highly purified yolk antibody simply, particularly highly purified IgY (Δ Fc), and the yolk antibody that is generated can be used in various immune purposes.
Therefore, one object of the present invention is to provide the method for selective separation IgY antibody in a kind of it from eggs of anseriformes Huang, it is characterized in that:
(a) the water insoluble and non-charged sorbent material with, the water that obtains from an Anseriformes birds yolk can adsorb yolk antibody in the miscible part isolate, this water insoluble and non-charged sorbent material is selected from the group that following material is formed: silicate, silicide, carbonate, vitriol, phosphoric acid salt, carbon, Mierocrystalline cellulose and synthon, pottery and metal oxide, and wherein the amount of this water insoluble and non-charged sorbent material is enough to can separate this yolk antibody in the miscible part isolate from this water; And
(b) flow through this water insoluble and non-charged sorbent material to obtain a water-based part isolate that comprises this yolk antibody with a damping fluid.
Another object of the present invention is to provide the method for selective separation IgY antibody in a kind of it from eggs of anseriformes Huang, it is characterized in that carrying out saltouing for the first time with the water-based part isolate that comprises yolk antibody of saltouing, it is with first kind of concentration (NH
4)
2SO
4Carry out, this first kind of range of concentrations is that the volume with this processed water-based part isolate is that benchmark is from about 15% (w/v) to about 24% (w/v), wherein be preferably and be not higher than about 21% (w/v), and processed this comprises the water-based part isolate of yolk antibody in saltouing for the first time to saltout then to carry out pickle change, and it is with second kind of concentration (NH
4)
2SO
4Carry out, this second kind of range of concentrations be the volume with this processed in saltouing for the first time water-based part isolate be benchmark from about 25% (w/v) to about 40% (w/v), wherein be preferably and be not higher than about 31% (w/v).
Another object of the present invention is to provide the method for selective separation IgY antibody in a kind of it from eggs of anseriformes Huang, it is characterized in that:
(a) the water insoluble and non-charged sorbent material with, the water that obtains from an Anseriformes birds yolk can adsorb yolk antibody in the miscible part isolate, this water insoluble and non-charged sorbent material system is selected from the group that following material is formed: silicate, silicide, carbonate, vitriol, phosphoric acid salt, carbon, Mierocrystalline cellulose and synthon, pottery and metal oxide, the amount of wherein should be water insoluble and non-charged sorbent material are enough to this yolk antibody of separation in this water can miscible part isolate;
(b) flow through this water insoluble and non-charged sorbent material to obtain a water-based part isolate that comprises this yolk antibody with a damping fluid;
(c) saltout the water-based part isolate that comprises yolk antibody in the step (b), it is with first kind of concentration (NH
4)
2SO
4Carry out, this first kind of range of concentrations be the volume with this processed water-based part isolate be benchmark from about 15% (w/v) to about 24% (w/v), wherein be preferably and be not higher than about 21% (w/v); And
(d) the processed water-based part isolate that comprises yolk antibody in step (c) of saltouing, it is with second kind of concentration (NH
4)
2SO
4Carry out, this second kind of range of concentrations be the volume with water-based part isolate processed in first saltouts be benchmark from about 25% (w/v) to about 40% (w/v), wherein be preferably and be not higher than about 31% (w/v).
According to the inventive method, can be used in selected isomer, particularly IgY (Δ Fc) antibody of a large amount of yolk antibodies of various industrial uses, can obtain in mode a kind of economy, efficient and that save time.
Another object of the present invention provide thus the IgY antibody made clinically with research on purposes.Except low-cost and preparation easily, the present invention's IgY (Δ Fc) antibody has for the Rheumatoid factors, polyclonal in complement system and the mammalian blood serum not tool activity and advantage that extremely low cross reaction is arranged for mammiferous IgG.Therefore, be specially adapted to relate to the immunoassay of the serum of Mammals, and have minimum interference.As is known to the person skilled in the art, the mode that IgY antibody can single agents exists and is used for clinical, academic research and other application, perhaps can be contained in the commercial kit, exists in the mode as an activeconstituents.
Another specific purpose of the present invention provides a kind of reagent that is used for an antigenic immunoassay of the cause of disease interested, and it comprises mat IgY antibody obtained by the method for the present invention.
Description of drawings
Above-mentioned and other purpose of the present invention and feature will become clearer by the narration of following preferred embodiment and the accompanying drawing of being followed, wherein:
Fig. 1 illustrates a relatively SDS-PAGE analysis of the antibody capture ability of four kinds of sorbent materials: the 1st swimming lane is through partially purified antibody extract; The 2nd swimming lane is the solution of 0.3% pyrogenic silica (fumed silica) of flowing through; The 3rd swimming lane is the solution of 3% silicon-dioxide of flowing through; The 4th swimming lane is the solution of 3%Celite diatomite of flowing through; The 5th swimming lane is the solution of 3%Celite diatomite hyflo-Cel of flowing through; With the 6th swimming lane be the solution of 5%Celite diatomite hyflo-Cel of flowing through;
Fig. 2 illustrates and utilizes pyrogenic silica to be used as the purified yolk antibody that goes out of sorbent material to carry out electrophoretic electrophoretic analysis result on the 8%SDS-polyacrylamide gel: the 1st swimming lane is through partially purified antibody extract; The 2nd swimming lane is the solution of 2% pyrogenic silica of flowing through; The 3rd swimming lane is the eluate that stems from this pyrogenic silica throw out; The 4th swimming lane is with 21% (w/v) the antibody product that ammonium sulfate was settled out in first settling step; The 5th swimming lane is with 31% (w/v) the antibody product that ammonium sulfate was settled out in second settling step;
Fig. 3 illustrates and utilizes Celite diatomite to be used as the purified yolk antibody that goes out of sorbent material to carry out electrophoretic electrophoretic analysis result on the 8%SDS-polyacrylamide gel: the 1st swimming lane is through partially purified antibody extract; The 2nd swimming lane is that Celite diatomite leaches thing; The 3rd swimming lane is with 21% (w/v) the antibody product that ammonium sulfate was settled out in first settling step; The 4th swimming lane is with 31% (w/v) the antibody product that ammonium sulfate was settled out in second settling step; The 5th swimming lane is with 16% (w/v) the antibody product that sodium sulfate was settled out in second settling step; And
Fig. 4 illustrates and utilizes pyrogenic silica to be used as the purified yolk antibody that goes out of sorbent material to carry out electrophoretic electrophoretic analysis result on the 8%SDS-polyacrylamide gel: M represents the molecular weight marker thing; The 1st swimming lane is through partially purified antibody extract; The 2nd swimming lane is the solution of 0.3% pyrogenic silica of flowing through; The 3rd swimming lane is the eluate that stems from this pyrogenic silica throw out; The 4th swimming lane is with 21% (w/v) the antibody product that ammonium sulfate was settled out; With the 5th swimming lane be through the purified antibody product that goes out of affinity chromatography.
Specific embodiments
In the ovum that birds serum and birds are produced, all be rich in yolk antibody.Yet as previously mentioned, it is normally preferred in considering of cost to collect this antibody by egg.Lay eggs hen with IgY and two kinds of isomers of IgY (Δ Fc) by serum transfers to yolk.In principle, contain IgY/ml and about IgY of 3 to about 12mg (Δ the Fc)/ml of the 1-4mg that has an appointment in the yolk of each duck's egg, therefore, in single egg, contain the antibody total amount and estimated and be about IgY of 15 to about 80mg and about IgY of 45 to about 240mg (Δ Fc).The yolk volume that is produced in any specific time quantitatively far surpasses the serum volume that obtains from these birds safely.In addition, the extraction of yolk antibody can be carried out on a large scale and not need a large amount of investments.Preferably, antibody of the present invention obtains from the Anseriformes birds egg with a specific antigen immunity.
According to the present invention, a kind of method that is used for isolating effectively from yolk antibody is provided, wherein so-called " adsorption chromatography " or " distinctiveness salting-out process " can be used alone or be used in combination with another person, the committed step when they are the conduct separation.
As used herein, " adsorption chromatography " speech is about a kind of separation method, and it comprises, and use one stationary phase optionally absorbs from a swimming mutually and the concentrated solute of being desired.According to one embodiment of the invention, one water insoluble and non-charged sorbent material in this stationary phase as activeconstituents, by yolk antibody being adsorbed in the water insoluble and non-charged sorbent material, and follow catch the water that is present in usually in the yolk can miscible lipid impurity, to separate this yolk antibody.
In embodiment preferred of the present invention,, then clean to remove egg white as far as possible with distilled water at first with this yolk and albumen sepn.Puncture the vitelline membrane (vitellinemembrane) that coats yolk, then should dilute with a large amount of aqueous buffer solutions or water by isolating yolk separate part, to form the suspended substance of a yolk.Collected yolk preferably with about 1: 2 to about 1: the aqueous buffer solution of the ratio of 40v/v or distilled water diluting, and more preferably with about 1: 5 to about 1: the ratio of 30v/v is diluted.Existing report points out that the pH value is the important factor (E.M.Akita and S.Nakai, J.Food Sci. the 57th volume, 629 pages (1993)) during the partial purification step.With regard to the optimum recovery rate of yolk antibody, pH preferably sets in about 5 between about 7 the scope.In this step, this temperature be more satisfactoryly about 0 ℃ between about 60 ℃ of scopes.The suspended substance that stirs this yolk carefully then leaves standstill one period that is enough to form water-based and non-aqueous layer to form the mixture of a homogeneous.Then remove water-insoluble substance by centrifugation in this water-based yolk suspended substance, it comprises non-aqueous biomolecules, such as lipoprotein, phospholipid, sterol and analogue thereof.The supernatant liquor that contains antibody that is then obtained can topple over, draw and other method and separating with this heavy-gravity throw out by known.
Generally speaking, the lipid composition of the water-soluble portion isolate that therefore obtains still Tai Gao so that harmful to follow-up operation.According to the present invention, one contains the stationary phase of water insoluble and non-charged sorbent material and water can the common incubation of miscible part isolate, and this sorbent material is one and is enough to adsorb and still is present in the consumption that the most of water of this water in can miscible part isolate can miscible lipid matter.The sorbent material that is fit to comprises but non-silicate, silicide, carbonate, vitriol, carbon, Mierocrystalline cellulose and synthon, pottery and the metal oxide of being confined to that wherein silicate comprises synthetic or natural clay, china clay, talcum and Calucium Silicate powder; Silicide comprises pyrogenic silica, non-crystalline silicon, silicon-dioxide, silica gel, silicate, diatomite and Fuller's earth; Carbonate comprises lime carbonate and barium carbonate; Vitriol comprises calcium sulfate; Phosphoric acid salt comprises calcium phosphate; Carbon comprises activated carbon and carbon fiber; Mierocrystalline cellulose and synthon comprise cellulose powder; Pottery comprises porous ceramics; And metal oxide comprises aluminum oxide and titanium oxide.
Particularly preferred sorbent material is pyrogenic silica, silicon-dioxide and diatomite.Sorbent material is decided on the characteristic of selected sorbent material the operation ratio that water can miscible part isolate, and can change in the scope of a broadness.When pyrogenic silica was used in this method, volume that can miscible part isolate with processed water was as the criterion, and is preferably and is added into the concentration that is equal to or higher than about 0.1 weight %, more preferably is positioned at about 0.3 scope to about 5.0 weight %.When sorbent material is silicon-dioxide or diatomite, adsorption chromatography according to the present invention is to be as the criterion at volume that can miscible part isolate with processed water, is preferably greater than about 1 weight % and more preferably be positioned under the sorbent material of about 3 to about 20 weight % scope and carry out.
According to the present invention, as long as it is gratifying being retained in the yolk antibody of adsorbent surface, this adsorption chromatography can be carried out by any known manner, such as can miscible part isolate with sorbent material batch processed water, or make water can miscible part separated stream through a chromatography column that is filled with sorbent material.Reaction times during the processing and temperature be for result and non-key, and about 4 to about 30 ℃ temperature of reaction and about 10 to about 60 minutes reaction times normally are suitable for.Single operation is promptly enough usually, and this adsorption step can be repeated for several times in the time of if necessary, and uses new sorbent material at every turn.By this flow process, fatty and most of non-fatty substance can successfully be separated into two immiscible phases, and yolk antibody also can be adsorbed.
Catch the ability of immunoglobulin (Ig) on selected sorbent material and decide, yolk antibody can be recovered from the eluate that gone out by stationary phase institute wash-out or " solution of flowing through ", and " solution of flowing through " that is used for this paper is the solution that means by stationary phase.Shown in the preferred embodiment that is provided in this article, when pyrogenic silica or silicon-dioxide were used as sorbent material, yolk antibody mainly was present in stationary phase, and diatomite is retained and surpassed about 60% antibody in the solution of flowing through.
Select specific method can decide by those skilled in the art with the wash-out yolk antibody.Typically, yolk antibody can be by flow through this water insoluble and non-charged sorbent material and in a water-based part isolate and get with a damping fluid, wherein this damping fluid is in and is lower than about 4 or be higher than about 8 pH value down or contain chaotropic agent and be available for the present invention, thereby by obtaining yolk antibody in the stationary phase, and can not make lipid break away from stationary phase substantially, so that one contains the antibody elution thing and is formed.This employed " eluate " speech system at one contain by eluent from stationary phase, wash from desire the solution of material.＂ chaotropic agent (chaotropic agent) ＂ or ＂ chaotropic agent (chaotrope) ＂ one speech are one can cause the chemical substance of the topographical variations of protein molecule (such as antibody molecule), and therefore, it is regarded as a protein denaturant usually.According to the present invention, most binding antibody all can be successfully contains gentle concentration (the neutral buffered liquid of＞1M) chaotropic agent comes wash-out with any.In most of examples, behind wash-out, remove this chaotropic agent and will reply natural protein structure.
The damping fluid that is suitable for includes but not limited to Repone K, the potassiumiodide of 5M, the magnesium chloride of 3.5M, thiocyanic acid ammonium salt/sodium salt/sylvite of 1-3M and the urea of 6M of 3M to 6M guanidine-HCl, 3.0M of 0.1M glycine-HCl, pH3.0 of 0.1M glycine-HCl, the pH10.0 of pH2.3.Yet, for the activity of antibody through reclaiming, one gentle ionic strength and contain the pH neutral damping fluid of chaotropic agent is such as through being buffered in 20mM MES damping fluid (pH5.8) or 20mM Tris (Tutofusin tris) 3M Sodium Thiocyanate 99 (pH7.5) preferably is used in enforcement the present invention.The active condition of collected antibody can be easily by replying such as a low ionic strength and a large amount of dialysis of not containing the weak acidic buffer of chaotropic agent.
According to one embodiment of the invention, this water-based part isolate comprises acceptance one distinctiveness that the eluate that is rich in antibody or the solution of flowing through the continues flow process of saltouing, to isolate the yolk antibody isomer.
In protein chemistry skill, present its common meaning at this " saltouing " speech that is used, its be at one or several non-sex change salt be added into a mixture or make in the batching, reducing protein solubility, and cause the precipitation or the cohesion of protein." distinctiveness is saltoutd " speech means a kind of salt concn that is added by variation, and is settled out to distinctiveness from a mixture or condenses two or more kinds of proteinic salting-out process.In the present invention, desire by distinctiveness the protein that is settled out be the yolk antibody isomer, i.e. IgY and IgY (Δ Fc).The example that is available for being settled out the non-sex change salt of yolk antibody comprises but the non-NaCl of being confined to, Na
2SO
4, (NH
4)
2SO
4, KCl, CaCl
2And MgSO
4Preferably, this non-sex change salt is Na
2SO
4Or (NH
4)
2SO
4, and especially with (NH
4)
2SO
4For good.The salt concn that is used for difference ground precipitation yolk antibody isomer is decided on the kind of this salt, and can be those skilled in the art and determine via simple test.According to a preferred embodiment of the invention, wherein use (NH
4)
2SO
4IgY is to be that benchmark is a scope from about 15% (w/v) to about 24% (w/v) at the processed volume with the eluate or the solution of flowing through, and be preferably under the salt concn that is equal to or less than about 21% (w/v) and at first salted out, and the processed volume that rises to the eluate or the solution of flowing through when this salt concn is that benchmark is that scope is from about 25% (w/v) to about 40% (w/v), and when being preferably about 31% (w/v), IgY (Δ Fc) is just precipitated to be gone out.Should understand the visual selected salt of the order of these two kinds of antibody isomer precipitations and changing.In this flow process, it also is feasible merging use two or more kinds of salt, for example, at first goes out first kind of isomer with a precipitation of salts, next goes out second isomer with another kind of precipitation of salts.According to the present invention, this distinctiveness flow process of saltouing is finished a main purpose of the present invention dramatically, that is, from by IgY and this two all yolk antibody of being formed of IgY (Δ Fc) substantially selective separation go out desirable IgY and comprise IgY and IgY (Δ Fc) antibody.
If it is desirable obtaining the antibody of a higher degree, can be dissolved in the suitable Laemmli buffer system Laemmli again through sedimentary antibody, and accept extra purifying flow process, such as size exclusion chromatography method, hydrophobic interaction chromatography method, ion exchange chromatography and immune-affinity chromatography.
As used herein, " immunoaffinity purification method " or terms such as " immune-affinity chromatographies " are based on the type of separation of this antibody to a special antigenic characterization of adsorption.This is to separate with unconjugated antibody under this condition in the antibody that is bonded to a specific antigen under the specified conditions promptly.The present invention is that the use with this immunoaffinity purification method removes uncorrelated protein, is non-antigen-binding immunoglobulin (Ig) especially.
According to the present invention, the immunoaffinity purification method is that the antigen matrix (antigen matrix) that application one is made up of the antigen that is fixed on the soluble support is carried out.The kind of this support is unessential for immunoaffinity purification method of the present invention.The nullvalent support material of interaction between any antibody that is applicable to an antigen covalent attachment traditionally and this institute is desired and the antigen that is fixed thereon all can be used.This support generally is made by crosslinked agarose or crosslinked dextran, such as this CNBr-activated agarose gel (Sepharose) 4B that can buy from Pharmacia.
Be dissolved in the binding buffer liquid by the purified antibody that goes out of distinctiveness salting-out process, and be applied on this antigen matrix, to make it to form the immunocomplex of immobilized antigen and yolk antibody.Any to this Ag-Ab the interaction Fails To Respond and can effectively keep the Laemmli buffer system Laemmli that this institute desires and all can be used for the present invention in conjunction with condition.This binding buffer liquid is preferably selected from the group that is made of phosphate buffered saline buffer, MES (2-[N-morpholine] ethyl sulfonic acid) damping fluid and bis-Tris damping fluid, is best at the MES of 20mM damping fluid wherein.
Preferably, this immunoaffinity purification is to carry out under the environment of weak acid and low ionic strength, that is, in the pH value that is positioned at about 4 to about 7 scope be lower than under the ionic strength of about 50mM.More preferably, this antibody with through fixed antigen about 5 to about 6 scope and most preferably be under about 5.6 the pH values to about 5.8 the scope and interact.Yolk antibody can or be lower than about 4 or be higher than under about 8 the pH value and break away from by chaotropic salt from antigen matrix.The activity of collected antibody can be by replying such as a low ionic strength and a large amount of dialysis of not containing the weak acidic buffer of chaotropic agent.
According to the purified IgY (Δ Fc) that goes out of the inventive method neither can the complement activation system can be in conjunction with the Rheumatoid factors, polyclonal of mammalian blood serum yet.Cross reaction between IgY (Δ Fc) and the mammiferous IgC is not remarkable.Therefore, the present invention also provides a kind of suitable confession to be used for novel antibody clinical and the research purposes.
The present invention also provides the various clinical of IgY antibody prepared in accordance with the present invention and research purposes.
For example, a kind of treatment or prevention animal (it comprises poultry, livestock, reaches pet) or patient's therapeutic composition is provided among the present invention, it comprises the IgY antibody of the present invention of a therapeutic dose, with protection or prevent its etc. be not subjected to influencing of various cause of disease material, this cause of disease material comprise microorganism, such as the synthetic virus of bacterium, natural or recombinant type or peptide, fungi, protozoon and nematode and analogue thereof; And the material of proteinic or nonprotein, can cause immunoreactive immunogen such as natural or recombinant type or peptide synthetic anaphylactogen, toxin, venom, hormone or any other.Purified IgY antibody preferably with a pharmaceutically acceptable supporting agent (such as, water, physiological saline etc.) combine and offerd medicine.This pharmaceutical composition can be oral, injection, external application and immunotherapy are thrown and given.
IgY antibody of the present invention also is available for detecting and stems from from the obtained body sample of the mankind or animal, interested cause of disease material such as body fluid, tissue, cell extract, it comprises the biology as pathogenic or non-virulent, such as intestinal bacteria (Escherichia coli), Salmonella enteritidis (Salmonella enterititis) and other bacillary biologies; One natural or recombinant type or peptide synthetic hormone, such as oestrogenic hormon, Progesterone, thyroxine and analogue thereof, main histocompatibility complex's antigen and analogue thereof; Natural or recombinant type or peptide synthetic tumor marker, such as α-fetoprotein, prostate gland specific antigen and analogue thereof, the morbid state marker is such as proteins C reactive matter, ferritin and analogue thereof; The accumulation of foreign matter or residual is such as theophylline and foxglove.For only obtaining at the special antibody of this cause of disease material, this cause of disease material of injectable as antigen, is desired production of antibodies to bring out in duck, and wherein this antigen comprises natural purifying antigen, recombinant antigen, peptide synthetic antigen and DNA plasmid.The IgY antibody that utilizes the present invention to obtain, cause of disease material interested can be by the traditional method of knowing in any this area, such as Ou Teleni (Ouchterlony) method, single radioimmunodiffusion (SRID), immunoelectrophoresis (IEP), radio immunoassay (RIA), enzyme labeled immunoassay adsorption analysis method (ELISA), Western blotting (WB), than turbid immunoassay (TIA), particle is promoted formula than turbid immunoassay, enzyme immunoassay, the turbidity measurement immunoassay, chemiluminescence immune assay, golden analytical method of immunity or immunochromatographiassays assays are quantitatively or qualitatively to detect.
IgY antibody of the present invention is also suitable for being used for biochip and biological inductor.
Followingly give purpose that example only is used for explanation but not intended the present invention's scope.
Embodiment 1: the immune programme for children that is used to stimulate the specific antibodies generation
The duck (Anasplatyrhynchos var.domestica) that is used to produce the artificial breeding of antibody and ovum for 12 16 week age is separated to feed.This duck is accepted the proteins C reactive matter (CRP of 1-5mg/ml; Purifying from the mankind's ascites and get) first subcutaneous injection, be formulated in proteins C reactive matter and the emulsification of equal-volume complete Freund's adjuvant in the phosphate buffered saline buffer of pH7.5.The antigenic concentration of using is generally between 1 to 5mg/ml the scope.After this initial injection, per fortnight of young hen is accepted the antigenic extra injection of 1-5mg, totally three times.After one week, begin to collect ovum, and mark and being stored under 4 ℃, up to the extraction of carrying out antibody and purifying.In experimental session, repeat to strengthen (booster) program per four week.The sampling of blood is carried out in after each booster shots the 7th day.The serum of centrifugal each blood sample and collection gained.
Embodiment 2: extract antibody by duck yolk
By the duck of the hyperimmunization among the embodiment 1 under ovum in and the yolk collected cleans with a weak distilled water water column fully, to remove egg white by this.Measure the volume of yolk, then mix fully with the distilled water of 10 times of amounts of measured yolk volume.Subsequently, mixture was preserved two hours down in 4 ℃ at least, subsequently with Hitachi CR22F whizzer in 10, under the 000rpm centrifugal one hour.Formation one light supernatant layer and half solid shape are subject to the disturbance layer in the centrifuge tube.
Embodiment 3: with sorbent treatment
In the prepared crude extract of embodiment 2, add following one of them sorbent material: 2% (w/v) pyrogenic silica (available from Sigma), 3% (w/v) silicon-dioxide (Sigma), 3% (w/v) Celite diatomite (available from Celite Corporation) and 3% or 5% (w/v) Celite diatomite hyflo-Cel (Celite Corporation).The suspension of gained is cultivated under 4 ℃ lasts 60 minutes, and imposes mild stirring.After finishing cultivation, under 4 ℃, in 20, under the 000rpm sorbent material is precipitated, and collected supernatant liquor and throw out respectively with the HitachiCR-22F whizzer.Get 10 μ l samples by each supernatant liquor and accept non-reduced SDS-polyacrylamide gel electrophoresis analysis (SDS-PAGE).
As shown in Figure 1, by the adsorbed antibody amount of sorbent material, pyrogenic silica has best adsorption activity, and does not almost have antibody to be retained in the solution of flowing through.Silicon-dioxide presents the avidity weak slightly to immunoglobulin (Ig), and this may be because it has lower porosity (and thereby comprise a small surface area) than pyrogenic silica.On the other hand, being lower than 10% yolk antibody is caught by the diatomite of arbitrary form.
Embodiment 4: the distinctiveness of yolk antibody is saltoutd
Table 1
| The total protein quality | IgY per-cent | IgY productive rate/ovum | IgY (Δ Fc) per-cent | IgY (Δ Fc) productive rate/ovum | |
| Crude extract | 447.4mg | ??4.43％ | 19.82mg | ??26.79％ | 119.86mg |
| Dash and carry thing | 224.45mg | ??8.15％ | 18.29mg | ??41.65％ | 93.48mg |
| Utilize 21% (NH 4) 2SO 4The 1st precipitation | 30.65mg | ??37.82％ | 11.59mg | ??62.18％ | 19.06mg |
| Utilize 31% (NH 4) 2SO 4The 2nd precipitation | 74.57mg | ??2.03％ | 1.51mg | ??96.62％ | 72.05mg |
As shown in table 1, gained IgY (Δ Fc) antibody is recovered with the purity that is higher than 96% with about 76% productive rate (72.05mg/119.86mg * 100%).Prior, this purifying flow process advantageously causes IgY (Δ Fc) antibody desired from substantially by separating the yolk antibody integral body (mainly being made up of IgY and IgY (Δ Fc) this two).
Embodiment 5: with Celite diatomite partial purification IgY (Δ Fc)
Utilize diatomite to attract lipid and repel the ability of antibody, will be in embodiment 2 prepared crude extract be poured into the volume that is filled with the crude extract of impouring and count in the diatomaceous Filter column of Celite of 10 weight %.The solution of this post of flowing through is collected, and makes it accept to count with the liquor capacity of flowing through the precipitation first time of 21% (w/v) ammonium sulfate.Be collected through sedimentary antibody, and with the supernatant liquor separated into two parts.The part of supernatant liquor is precipitated with the ammonium sulfate of the concentration of about 31% (w/v), and another part is then with the sodium sulfate precipitation of the concentration of 16% (w/v).The antibody product that is settled out is dissolved among the PBS again.In 8% irreducibility acrylamide gel, implement analytical SDS-PAGE, wherein be written into the crude extract (the 1st swimming lane) of 2012.5 μ g embodiment 2, the solution of flowing through (the 2nd swimming lane) that 1678 μ g are obtained by the Celite diatomite filtration, 94.9 μ g at the resulting eluate of first settling step (the 3rd swimming lane) and 169.65 μ g and 357.75 μ g by 31% ammonium sulfate and 16% sodium sulfate at the resulting antibody product of second settling step (the 4th, 5 swimming lane).The result as shown in Figure 3.Reclaim per-cent and purity and measure, and be summarized in the table 2 by the scanning intensity of this gel.
Table 2
| The total protein quality | IgY per-cent | IgY productive rate/ovum | IgY (Δ Fc) per-cent | IgY (Δ Fc) productive rate/ovum | |
| Crude extract | 405.2mg | ?11.60％ | 46.98mg | 29.01％ | 117mg |
| CLT filters | 335.6mg | ?5.08％ | 17.05mg | 30.47％ | 102.25mg |
| Utilize 21% (NH 4) 2SO 4The 1st precipitation | 18.98mg | ?62.60％ | 11.88mg | 37.40％ | 7.10mg |
| Utilize 16% Na 2SO 4The 2nd precipitation | 33.93mg | ?6.29％ | 2.13mg | 77.14％ | 26.17mg |
| Utilize 31% (NH 4) 2SO 4The 2nd precipitation | 71.55mg | ?8.79％ | 6.29mg | 68.68％ | 49.14mg |
As shown in table 2, the IgY of gained (Δ Fc) antibody is recovered with the purity (when ammonium sulfate is used in second settling step) of about 77% (when sodium sulfate is used in second settling step) and 69% respectively, and all has high yield.
Embodiment 6: the immunoaffinity purification of yolk antibody
Preparation CRP solution in the 0.1M carbonate buffer solution, pH is 8.5, and concentration is 5mg/ml.Just begun 1mM cryosel acid elution available from CNBr-activated agarose gel (Sepharose) 4B of Pharmacia, then made it to spend the night with the CRP solution reactions of 2 times of amounts of matrix volume down in 4 ℃ to be 10 times of amounts of this matrix volume.This antigen matrix is suspended in the thanomin that is formulated in the 0.5M among the 20mM Tris-HCl (pH 8.5) with the ratio of 1: 1 (v/v) under 4 ℃, lasts 2 hours, the protein action site that still exists with blocking-up.Then, antigen matrix is cleaned with the PBS that contains 0.02% sodiumazide, and be stored in 4 ℃.
Use in embodiment 4 with 21% (w/v) the duck antibody that ammonium sulfate was settled out with by above-mentioned prepared antigen matrix.The antigen matrix of 1ml is loaded in a conventional post, and is soaked in 20mM MES (2-[N-morpholine] ethyl sulfonic acid) damping fluid (pH5.8).The antibody that this antigen matrix and 0.25ml are formulated in the identical combination damping fluid reacts.This antigen matrix is cleaned with this binding buffer liquid, till effluent does not contain protein in fact.Institute's bonded antibody is immediately with 6M guanidine-HCl wash-out in addition, and after dialysis fully, measures its optical density (OD) down in 280nm.Point out that in SDS-PAGE analysis shown in Figure 4 mainly be made up of IgY (Δ Fc) antibody through the antibody of affinity purification, it is to present single bands of a spectrum on gel.
The all patents quoted from the present invention's the specification sheets and bibliographic reference data are incorporated this paper into as the reference data in full with it.If with this case the conflict part is arranged, then the explanation (comprising definition) with the present invention is as the criterion.
Though the present invention has also utilized above-mentioned specific embodiment to be illustrated, it must be appreciated that showing various improvement and the variation known to those skilled in the art can carry out under spirit that does not depart from the present invention and category.
Claims (35)
1. the method for selective separation IgY antibody in the it from eggs of anseriformes Huang is characterized in that:
(a) the water insoluble and non-charged sorbent material with, the water that obtains from an Anseriformes birds yolk can adsorb yolk antibody in the miscible part isolate, this water insoluble and non-charged sorbent material is selected from the group that following material is formed: silicate, silicide, carbonate, vitriol, phosphoric acid salt, carbon, Mierocrystalline cellulose and synthon, pottery and metal oxide, and wherein the amount of this water insoluble and non-charged sorbent material is enough to can separate this yolk antibody in the miscible part isolate from this water; And
(b) flow through this water insoluble and non-charged sorbent material to obtain a water-based part isolate that comprises this yolk antibody with a damping fluid.
2. according to the process of claim 1 wherein that silicate preferably includes synthetic or natural clay, china clay, talcum and Calucium Silicate powder; Silicide preferably includes pyrogenic silica, non-crystalline silicon, silicon-dioxide, silica gel, silicate, diatomite and Fuller's earth; Carbonate preferably includes lime carbonate and barium carbonate; Vitriol preferably includes calcium sulfate; Phosphoric acid salt preferably includes calcium phosphate; Carbon preferably includes activated carbon and carbon fiber; Mierocrystalline cellulose and synthon preferably include cellulose powder; Pottery preferably includes porous ceramics; And metal oxide preferably includes aluminum oxide and titanium oxide.
3. according to the process of claim 1 wherein that these Anseriformes birds are duck or goose.
4. according to the method for claim 1, be used to produce antibody.
5. according to the process of claim 1 wherein that be used in the step (b) the to flow through damping fluid of this water insoluble and non-charged sorbent material comprises a wash-out salt, wherein preferably, this wash-out salt comprises about 3M to the Guanidinium hydrochloride of about 6M or the about 1M Sodium Thiocyanate 99 of about 3M extremely.
6. according to the method for claim 1, further comprise a purification step, it is to be about 4 to about 7 with immune-affinity chromatography in pH value scope, and preferred pH value scope is about 5 to about 6, and more preferably pH value scope is about 5.6 to about 5.8 and is lower than under the ionic strength of about 50mM and carries out.
7. according to the process of claim 1 wherein that this water-based part isolate that comprises this yolk antibody is the solution of flowing through in the step (b), its this water insoluble and non-charged sorbent material of flowing through.
8. according to the process of claim 1 wherein that this water-based part isolate that comprises this yolk antibody is an eluate in the step (b), it is a wash-out and going out in this water insoluble and non-charged sorbent material.
9. according to the process of claim 1 wherein that this IgY antibody comprises the antibody with Fc zone and reaches the antibody that does not have the Fc zone.
10. the method for selective separation IgY antibody in the it from eggs of anseriformes Huang is characterized in that carrying out saltouing for the first time with the water-based part isolate that comprises yolk antibody of saltouing, and it is with first kind of concentration (NH
4)
2SO
4Carry out, this first kind of range of concentrations is that the volume with this processed water-based part isolate is that benchmark is from about 15% (w/v) to about 24% (w/v), be preferably and be not higher than about 21% (w/v), and processed this comprises the water-based part isolate of yolk antibody in saltouing for the first time to saltout then to carry out pickle change, and it is with second kind of concentration (NH
4)
2SO
4Carry out, this second kind of range of concentrations is that the volume with this processed in saltouing for the first time water-based part isolate is that benchmark is from about 25% (w/v) to about 40% (w/v), is preferably not to be higher than about 31% (w/v).
11. according to the method for claim 10, wherein these Anseriformes birds are duck or goose.
12., be used to produce antibody according to the method for claim 10.
13. method according to claim 10, this damping fluid of this water insoluble and non-charged sorbent material of wherein being used in the step (b) to flow through comprises a wash-out salt, wherein preferably, this wash-out salt comprises about 3M to the Guanidinium hydrochloride of about 6M or the about 1M Sodium Thiocyanate 99 of about 3M extremely.
14. according to the method for claim 10, wherein this IgY antibody comprises antibody with Fc zone and does not have the antibody in Fc zone.
15. the method for selective separation IgY antibody in the it from eggs of anseriformes Huang is characterized in that:
(a) the water insoluble and non-charged sorbent material with, a water that obtains from an Anseriformes birds yolk can adsorb yolk antibody in the miscible part isolate, this water insoluble and non-charged sorbent material system is selected from the group that following material is formed: silicate, silicide, carbonate, vitriol, phosphoric acid salt, carbon, Mierocrystalline cellulose and synthon, pottery and metal oxide, the amount of wherein should be water insoluble and non-charged sorbent material are enough to this yolk antibody of separation in this water can miscible part isolate;
(b) flow through this water insoluble and non-charged sorbent material to obtain a water-based part isolate that comprises this yolk antibody with a damping fluid;
(c) saltout in the step (b) that this comprises the water-based part isolate of yolk antibody, it is with first kind of concentration (NH
4)
2SO
4Carry out, this first kind of range of concentrations is that the volume with this processed water-based part isolate is that benchmark is from about 15% (w/v) to about 24% (w/v), is preferably not to be higher than about 21% (w/v); And
(d) saltout that processed this comprises the water-based part isolate of yolk antibody in step (c), it is with second kind of concentration (NH
4)
2SO4 carries out, and this second kind of range of concentrations is that the volume with this processed in saltouing for the first time water-based part isolate is that benchmark is from about 25% (w/v) to about 40% (w/v), is preferably not to be higher than about 31% (w/v).
16. according to the method for claim 15, wherein silicate preferably includes synthetic or natural clay, china clay, talcum and Calucium Silicate powder; Silicide preferably includes pyrogenic silica, non-crystalline silicon, silicon-dioxide, silica gel, silicate, diatomite and Fuller's earth; Carbonate preferably includes lime carbonate and barium carbonate; Vitriol preferably includes calcium sulfate; Phosphoric acid salt preferably includes calcium phosphate; Carbon preferably includes activated carbon and carbon fiber; Mierocrystalline cellulose and synthon preferably include cellulose powder; The pottery bag preferably includes porous ceramics; And metal oxide preferably includes aluminum oxide and titanium oxide.
17. according to the method for claim 15, wherein these Anseriformes birds are duck or goose.
18., be used to produce antibody according to the method for claim 15.
19. according to the method for claim 15, this damping fluid of this water insoluble and non-charged sorbent material that wherein is used to flow through in step (b) comprises a wash-out salt, preferably, this wash-out salt comprises about 3M to the Guanidinium hydrochloride of about 6M or the about 1M Sodium Thiocyanate 99 of about 3M extremely.
20. according to the method for claim 15, further comprise a purification step, it is to be about 4 to about 7 with immune-affinity chromatography in pH value scope, preferably, pH value scope is about 5 to about 6, and more preferably, pH value scope is about 5.6 to about 5.8 and is lower than under the ionic strength of about 50mM and carries out.
21. according to the method for claim 15, wherein this water-based part isolate that comprises this yolk antibody is the solution of flowing through in the step (b), its this water insoluble and non-charged sorbent material of flowing through.
22. according to the method for claim 15, wherein this water-based part isolate that comprises this yolk antibody is an eluate in the step (b), it is a wash-out and going out in this water insoluble and non-charged sorbent material.
23. according to the method for claim 15, wherein this IgY antibody comprises antibody with Fc zone and does not have the antibody in Fc zone.
24. the IgY antibody that selective separation goes out in the it from eggs of anseriformes Huang, it is that this method feature is with method preparation:
(a) the water insoluble and non-charged sorbent material with, a water that obtains from an Anseriformes birds yolk can adsorb yolk antibody in the miscible part isolate, this water insoluble and non-charged sorbent material system is selected from the group that following material is formed: silicate, silicide, carbonate, vitriol, phosphoric acid salt, carbon, Mierocrystalline cellulose and synthon, pottery and metal oxide, the amount of wherein should be water insoluble and non-charged sorbent material are enough to this yolk antibody of separation in this water can miscible part isolate;
(b) flow through this water insoluble and non-charged sorbent material to obtain a water-based part isolate that comprises this yolk antibody with a damping fluid;
(c) saltout in the step (b) that this comprises the water-based part isolate of yolk antibody, it is with first kind of concentration (NH
4)
2SO
4Carry out, this first kind of range of concentrations is that the volume with this processed water-based part isolate is that benchmark is from about 15% (w/v) to about 24% (w/v), is preferably not to be higher than about 21% (w/v); And
(d) saltout that processed this comprises the water-based part isolate of yolk antibody in step (c), it is with second kind of concentration (NH
4)
28O
4Carry out, this second kind of range of concentrations is that the volume with this processed in first saltouts water-based part isolate is that benchmark is from about 25% (w/v) to about 40% (w/v), is preferably not to be higher than about 31% (w/v).
25. according to the antibody of claim 24, wherein step (a) mesosilicic acid salt preferably includes synthetic or natural clay, china clay, talcum and Calucium Silicate powder; Silicide preferably includes pyrogenic silica, non-crystalline silicon, silicon-dioxide, silica gel, silicate, diatomite and Fuller's earth; Carbonate preferably includes lime carbonate and barium carbonate; Vitriol preferably includes calcium sulfate; Phosphoric acid salt preferably includes calcium phosphate; Carbon preferably includes activated carbon and carbon fiber; Mierocrystalline cellulose and synthon preferably include cellulose powder; Pottery preferably includes porous ceramics; And metal oxide preferably includes aluminum oxide and titanium oxide.
26. according to the IgY antibody of claim 24, wherein these Anseriformes birds are duck or goose.
27. IgY antibody according to claim 24, this damping fluid of this water insoluble and non-charged sorbent material of wherein being used to flow through in step (b) comprises a chaotropic salt, preferably, this chaotropic salt comprises about 3M to the Guanidinium hydrochloride of about 6M or the about 1M Sodium Thiocyanate 99 of about 3M extremely.
28. the IgY antibody according to claim 24 further comprises a purification step, it is to be about 4 to about 7 with immune-affinity chromatography in pH value scope, preferably, pH value scope is about 5 to about 6, and more preferably, pH value scope is about 5.6 to about 5.8 and is lower than under the ionic strength of about 50mM and carries out.
29. according to the IgY antibody of claim 24, wherein this water-based part isolate that comprises this yolk antibody is the solution of flowing through in the step (b), its this water insoluble and non-charged sorbent material of flowing through.
30. according to the IgY antibody of claim 24, wherein this water-based part isolate that comprises this yolk antibody is an eluate in the step (b), its wash-out and going out in this water insoluble and non-charged sorbent material.
31., comprise that the antibody with Fc zone reaches the antibody that does not have the Fc zone according to the IgY antibody of claim 24.
32. a pharmaceutical composition, it comprises according to the IgY antibody of claim 24 and a pharmaceutically acceptable supporting agent, and preferably, this pharmaceutical composition is used for the treatment of an individuality.
33. according to the pharmaceutical composition of claim 32, wherein this IgY antibody comprises that the antibody with Fc zone reaches the antibody that does not have the Fc zone.
34. a test kit that is used for immunoassay, it comprises the IgY antibody according to claim 24.
35. according to the test kit of claim 34, wherein this IgY antibody comprises that the antibody with Fc zone reaches the antibody that does not have the Fc zone.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021232377A CN100355785C (en) | 2002-06-12 | 2002-06-12 | Process for selectively separating IgY antibody from anseriforme birds eggs and IgY antibody produced therefrom |
| CN200610128846A CN100591694C (en) | 2002-06-12 | 2002-06-12 | Igy antibody and method for selectively separating it from eggs of anseriformes |
| HK04104388A HK1061567A1 (en) | 2002-06-12 | 2004-06-16 | Process for selectively isolating igy antibodies from egg yolk of an anseriform bird and igy antibodies obtained thereby |
| HK07110599.7A HK1102410A1 (en) | 2002-06-12 | 2004-06-16 | Process for selectively isolating igy antibodies from egg yolk of an anseriform bird and igy antibodies obtained thereby |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021232377A CN100355785C (en) | 2002-06-12 | 2002-06-12 | Process for selectively separating IgY antibody from anseriforme birds eggs and IgY antibody produced therefrom |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200610128846A Division CN100591694C (en) | 2002-06-12 | 2002-06-12 | Igy antibody and method for selectively separating it from eggs of anseriformes |
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|---|---|
| CN1463985A true CN1463985A (en) | 2003-12-31 |
| CN100355785C CN100355785C (en) | 2007-12-19 |
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|---|---|---|---|
| CN200610128846A Expired - Lifetime CN100591694C (en) | 2002-06-12 | 2002-06-12 | Igy antibody and method for selectively separating it from eggs of anseriformes |
| CNB021232377A Expired - Lifetime CN100355785C (en) | 2002-06-12 | 2002-06-12 | Process for selectively separating IgY antibody from anseriforme birds eggs and IgY antibody produced therefrom |
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| CN200610128846A Expired - Lifetime CN100591694C (en) | 2002-06-12 | 2002-06-12 | Igy antibody and method for selectively separating it from eggs of anseriformes |
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| HK (2) | HK1102410A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101048426A (en) * | 2004-10-22 | 2007-10-03 | 金克克国际有限公司 | Isolating human antibodies |
| CN100434119C (en) * | 2006-09-04 | 2008-11-19 | 杨建彬 | Method for producing yelk antibody liquid in high immunity |
| CN109313181A (en) * | 2016-06-02 | 2019-02-05 | 朋友股份有限公司 | The antigen of egg allergy |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103910800A (en) * | 2014-04-09 | 2014-07-09 | 杭州雅盛生物科技有限公司 | Anti-HER2 polyclonal antibody as well as preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3045229C2 (en) * | 1980-12-01 | 1982-12-02 | Deutsche Texaco Ag, 2000 Hamburg | Process for the production of vinylcyclohexene |
-
2002
- 2002-06-12 CN CN200610128846A patent/CN100591694C/en not_active Expired - Lifetime
- 2002-06-12 CN CNB021232377A patent/CN100355785C/en not_active Expired - Lifetime
-
2004
- 2004-06-16 HK HK07110599.7A patent/HK1102410A1/en not_active IP Right Cessation
- 2004-06-16 HK HK04104388A patent/HK1061567A1/en not_active IP Right Cessation
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101048426A (en) * | 2004-10-22 | 2007-10-03 | 金克克国际有限公司 | Isolating human antibodies |
| CN100434119C (en) * | 2006-09-04 | 2008-11-19 | 杨建彬 | Method for producing yelk antibody liquid in high immunity |
| CN109313181A (en) * | 2016-06-02 | 2019-02-05 | 朋友股份有限公司 | The antigen of egg allergy |
| CN109313181B (en) * | 2016-06-02 | 2022-04-12 | 朋友股份有限公司 | Antigens of egg allergy |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1939931A (en) | 2007-04-04 |
| HK1102410A1 (en) | 2007-11-23 |
| CN100355785C (en) | 2007-12-19 |
| CN100591694C (en) | 2010-02-24 |
| HK1061567A1 (en) | 2004-09-24 |
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