TW587941B - Manufacture of IgY(DeltaFc) antibodies and uses thereof - Google Patents

Abstract

The present invention relates to a method for the preparation and purification of IgY(DeltaFc) antibody from avian yolk, generally comprising the steps of immunization of a fowl hen with an antigen, a partial purification of the whole antibodies from the eggs laid by the hen, and an immunoaffinity purification of the antibodies raised against the antigen, in which the binding of the antibodies with the antigen in the immunoaffinity purification step is conducted at pH within a range of 4-7 and under an ionic strength of lower than 50 mM. The present invention also relates to the IgY(DeltaFc) antibody produced thereby and various uses of the novel IgY(DeltaFc) antibody.

Description

587941 A7 ________ B7 V. Description of the invention (1) Background of the invention 1) The scope of the invention (please read the notes on the back before filling this page) The invention relates to a method for preparing and purifying IgY (A Fc ) Method of antibodies, and IgY (A Fc) antibodies produced by this method. The invention also relates to the use of this novel IgY (A Fc) antibody for quantitative or qualitative analysis of a causative antigen of interest. 2) Explanation of related techniques Anti-system has been widely used in a variety of biological research and clinical applications. Sera obtained from highly immune mammals are the most common source of multiple antibodies. The immune system obtained from such immune sera belongs to a group of proteins generally referred to as "immunoglobulins", and among them, immunoglobulin G (I g G) is the largest. The I g molecule It consists of three domains, including two Fab regions and one Fc region. The Fab site is mainly involved in antigen binding. Although the Fc site does not have the ability to bind to the antigen, it is responsible for the management of an antibody. Various biological activities, such as complement fixing and Fc receptor binding. The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed on the technology of immunodiagnostics. A complete Ig G molecule is not suitable for mammals. In the detection system and immunoassay of jk Qing, it is because the Fc region on the IgG molecule has binding to the Fc receptor, activates the complement system, and rheumatoid factors in mammalian serum. Response, etc. Removal of the Fc portion of an IgG molecule often leads to a reduction in such interference (E. Lamoyi et al., 1986, "Methods in Enzymology ) ", Vol. 121, pp. 652-663). Page 3 This paper size applies the Chinese National Standard (CNS) A4 (210X297 mm) 587941 A7 B7 V. Description of the invention (2) Antibodies for immunotherapy Some of the recommended uses include the treatment of patients with virulent bacterial toxins or snake venom (eg, US Patent Nos. 5,340,923 and 5,601,823), and the prevention of lethal enterocoliosis in newborn piglets (H. Brussow et al., J. Ciin. Microbiol. Vol. 25, p. 982 (1987); and C.〇.Tacket et al., New Eng. J.

Med. Vol. 318, p. 1240 (1 988)). The Fc segment of an antibody molecule is known to be the most antigenic part of the immunoglobulin (E.M. Akita et al. J Immunol. Methods. Vol. 162, 1 55-164 (1 993)), Bay 1J J: Resection of the dagger segment (which will cause the formation of the F (ab ') 2 segment) will significantly reduce the potential allergen locations that are mostly located on the immunoglobulin molecule. Human or animal is beneficial. It has recently been shown that the double-bonded F (ab'h antibody segment is more suitable for this immunodiagnostic test (M. Mumsugu et al., J. COilc) ld Intei.faee Sq Vol. ⑷, p. 378 ( 1991); and j. L. Ortega_Vinuesa #, J. Immunol Methods Vol. 90, p. 29 (1996)), and is more suitable than itself for the development of immunoassays involving mammalian serum. However, 'the F (ab, h antibody segment is not widely used in clinical immunodiagnostic kits as expected by the general public. It may be attributed to the f (printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, Ministry of Economic Affairs, China) (Please read the precautions on the back before filling this page) Φ0! The difficulty of mass production and the reasons for not meeting economic efficiency, section

The F (ab ') 2 segment is traditionally prepared by the hydrolysis of IgG through gastric protein and A, and then purification by chromatography. Ducks and their taxonomic relationships are similar. M and σ 卩 reptiles (such as turtles) have three types of serum immunoglobulins: a knife immunoglobulin

IgM (800 kDa in ducks), and the sedimentation coefficient of each of the two species is' M (Ducks, page 4) The paper size applies the Chinese National Standard (CNS) A4 specification (210 × 297 public envy) 587941 A7 ______ B7 V. Description of invention (3 180 kDa in) and 5.7S (130 kDa in duck)]; isomeric form of gG (ER Unanue et al., J. Exp. Med. Vol. 121, (Please read the notes on the back first Refill this page} 697-714 (1965); Η · M. Grey, J. Immunol Vol. 98, 811-819

(1967); and B. Zimmerman et al., Biochemistry Vol. 10, pp. 482-44 8 (197 1)). Avian IgG is generally called Ig γ because it exists in the egg's egg '. The 5.7S IgY composed of a shorter heavy chain is similar in structure and antigenicity to the F (ab,) 2 segment of the 7.8S IgY (as shown in Figure 1). This fact leads to the use of IgY ( Equivalent to 7.8S IgY) and IgY (AFc) (equivalent to 5.7S IgY) are named to represent the two isomeric forms of IgY (K · E. Magor et al., J. Immun〇i · Vol. 149, 2627- 2633 (1992)). Research conducted by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs printed on infected or experimentally immunized birds shows that the duck resistance system lacks most biological effector functions, including complement binding and Fc receptor binding, but without sacrificing Their binding activity to the corresponding antigens (GW Litman Search, Immunochemistry Vol. 10, 323 (1973); and T. E. Toth et al., Avian Dis. Vol. 25, 17-28 (1981)) . It may be due to the apparent absence of the FC-equivalent region of the ι2γ (Δ Fc) antibody ' The I g Y (Δ F c) antibody system constitutes a major part of the duck antibody response. Therefore, it is believed that if a reliable method for manufacturing the antibody can be found 'and the physical elements suitable for its activity can be identified, then the IgY (A FC) antibodies will provide extraordinary benefits in immunological applications. It has been reported that avian yolk resistance systems, like mammalian antibodies, have properties useful for research and clinical applications. (For example, page 5 of the US Patent No. This paper is applicable to the Chinese National Standard (CNS) M specification (2 丨 0 > < 297 mm) 587941 A7 B7 V. Description of the invention (4) 5,340,923; US 5,5 85,098; US 5,60 1,823; and U: S. 5,976,5 1 9). The yolk line obtained from an egg-laying female is cheaper and more convenient and safer to operate than highly immune mammalian serum. More importantly, flavin antibodies can be adapted to the relevant legal norms of modern animal protection (A. Poison et al., Immunol. Commun. Vol. 9, p. 475 (1 980); and B. Gottstein et al.). These facts suggest that egg yolk has the potential to be used as a commercial source of antibodies. At present, efforts have been made to isolate and purify IgY from yolk. Examples include agar, viscose (in Japanese Laid-Open Publication No. 64-38098 published on February 8, 1989), dextran sulfate (JC Jensenius et al., J. Immunol. Methods vol. 46, p. 63 (1 98 1)), natural gums (H. Hatta et al., J. Food Science Vol. 53, p. 425 (1988)) and polyethylene glycol (PEG) (A. Poison et al. , Immunol. Invest .. Vol. 14, page 323 (1 985); can also be found in US Patent No. 4,550,019 issued to A. Poison and other substances are used to precipitate non-aqueous biomolecules, mainly lipids And yolk particles to collect a water-soluble phase rich in yolk antibodies. A. Hass 1 et al. Developed a two-step chromatography method that includes hydrophobic parent interaction chromatography and size difference chromatography to further isolate the yolk antibody from a PEG-purified fraction ( a. Hassl and H Aspock, J. Immunol. Methods Vol. 110, p. 225 (1 988)). Akita et al. Have described an improved method for isolating igY 'wherein the yolk antibody system dilutes the yolk with a large volume of water, and then the obtained supernatant is subjected to size difference chromatography and / or an ion exchange layer It is obtained by extraction from eggs (EM Akita et al., J., page 6) The paper size applies to Chinese National Standard (CNS) A4 (210XM? Mm) (Please read the precautions on the back before filling in this page)

Order Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 587941 Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the Invention (5)

Immunol. Methods, vols. 160, 207 (1 993); and E. M. Akita and S. Nakai, J. Food Sci. Vol. 57, 629 (1 993)). However, all these studies and patents have focused only on the isolation of whole yolk antibodies (which includes at least IgY and IgY (Δ Fc)) from bird yolks, and not on the isolation of individual igY (A Fc) antibodies. Furthermore, since the IgY (AFc) resistance system exists only in birds (such as ducks and geese) of the genus Orcjer Anseriformes, it is performed on galllfom birds (such as chickens and turkeys). The isolation method cannot be reminiscent of the successful purification of an IgY (A Fc) antibody. In 1989, Higgins tried to prepare these antibodies from highly immune duck serum ', but the antibodies that were affinity purified at pH 8.0 and 0.5 M NaCl were not able to exert an effective precipitation reaction or aggregation reaction (D. Α Hlggins, Comp. Biochem. Physiol. Vol. 93B, pp. 135-144 (1.989)). As claimed by Hlggins in the literature, the optimal pH for the formation of duck antibody precipitin is in the range of pH 8.5 to pH 9.05. Since then, no important studies have been published on the isolation of IgY (A Fc) antibodies and their possible use. Therefore, there is a need in the art for a fast, low-cost, and efficient method. This method can easily isolate the desired IgY (AFc) antibodies from all antibodies while maintaining their activity. Furthermore, there is a need in the art for substantially purified IgY (AFc), which can be used as a new type of F (ab, h antibody for various immunodiagnostics and immunotherapy.) Overview Page 7 This paper size applies Chinese National Standard (CNS) A4 size (210x297 male sauce) ----- (Please read the precautions on the back before filling this page)

587941, invention description (6) I have conducted extensive research to meet the needs of Shangcheng and the industry. Surprisingly, we found that the production of a ^ 1 egg 5 antibody ", a successful method for the isolation of antibodies to the dragon and your dragon, the interaction between the antigen and the antibody during the 1gY (A Fc) step of the steam engine \ The "harmonic purification step" is easily achieved through a simple procedure. Under the combined conditions, from today +

With economic benefits, a method that can produce F (ab,) 2 antibodies with a month is simply produced, such as the purified one. A new type of Fc) anti-system produced by sacrifice can be used in various immunization applications and the produced IgY (A). Therefore, an object of the present invention is to provide a method for preparing an IgY (A Fc) antibody in an egg. This method A step born from a female bird: Immunizing the poultry's beach food with an immunogen // Lambda contains the following steps # 小 宵, purification and purification of whole antibodies from female gold &, and combating the history # ^ ^ t Immunoaffinity purification of antibodies produced by the antigen, in which the binding of the antibody to the immunogen / antigen is performed in a weak acid and low ionic strength environment in the immunoaffinity, heptogenization steps. Especially, The antibody-antigen interaction is performed at an ionic strength of less than 50 mM 'in the range of pH 4-7 to obtain an optimal result. Therefore, the present invention provides a method for self-yolk A method for preparing IgY (A Fc), comprising the following steps: (a) Immunizing a female poultry with a selected antigen, so that pheasant antibodies raised by the antigen can be accumulated in the yellow print; (b) collecting The yolk of the immunized female is removed, and the nutrient water Molecules and particles to obtain a water-soluble separation fraction containing egg yolk antibodies; (c) at an ionic strength of less than 50 mM, in the range of pH 4-7. Standard (CNS) A4 specification (210 > < 297 public directors) (Please read the precautions on the back before filling this page)

-Order- Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 587941

5. Description of the invention (internally "let the water-soluble separation portion pass through an inert carrier matrix having the antigen immobilized thereon" to allow the formation of an immune complex of the immobilized antigen and the yolk antibody; and (d) from the The yolk antibody is isolated and extracted from the immobilized antigen. Another object of the present invention is to provide a novel IgY (A Fc) antibody produced by the method of the present invention. Another object of the present invention is to generate the IgY produced by the method. (A Fc) antibodies are provided for clinical and research applications. In addition to low cost and easy preparation, the IgY (A? Anti-hook system of the present invention has the advantages of not activating the complement system and rheumatoid factor in the serum of mammals, At the same time, it has adverse interactions with mammalian IgG, so it is particularly suitable for immunoassay involving mammalian sera with minimal interference. As those skilled in the art know, the antibody can exist as a single reagent. It can be used in clinical, academic research, and other applications, or it can be included in a commercial set as an active ingredient. Another aspect of the present invention A special purpose is to provide a reagent for immunoassay of a causative substance of interest, which comprises an IgY (A Fc) antibody prepared by the method of the present invention. Another object of the present invention is to provide an immunity A method of analysis and a commercial kit for performing the immunoassay, in which an antibody against a causative substance (preferably an IgY (A Fc) antibody purified by the present invention) is bound to the causative substance Under the optimal conditions, culture in the presence or absence of the causative substance of interest to quantitatively or qualitatively analyze the causative substance. According to the present invention, the optimal condition is an ion below 50 m Μ. Under the conditions of strength, the pH is in the range of 4-7. '' Page 9 This paper size applies the Chinese National Standard (CNS) M specifications (2 丨 0 > < 297 public shame) (Please read the precautions on the back before filling (This page)

, 1T ««: Printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 587941 V. Description of the Invention And the accompanying narrative of the diagrams became more clear. The better of Ju 1 is Jujui, and the figure 1 is a schematic diagram showing a typical primate and / or heterogeneous form. Duck IgY Figure 2 shows the state of the two SDS-polyacrylamide gel electrophoresis prepared in the present invention, and the state of anti- (Co⑽assie Blue) staining: 1 in Comexi blue; the crude Extracted antibody extracts in the second runway, the first runway immunoaffinity chromatography flow through the substance in the third runway = = affinity purification of antibody passages in the fourth runway; and Yu Chong The third figure is a graph showing the reactivity (mg / dl, logarithmic scale) of C · reactive protein (the diameter of the precipitation circle of ⑽ corresponds to the serobar quasi σ ·, .goat inverse Θ 铩 sheep σα; Fig. 4 shows the Peipei degree-bile analysis according to the present invention And—comparison of CRp concentrations obtained by a commercial immunoturbidimetric analysis. Figure 5 illustrates an ELISA analysis in which the ㈣ △ &) antibody system of the present invention is used to determine the CRP concentration, and Section 6a The -b diagram illustrates a particle-promoting turbidimetric immunoassay in which the secret Fc) anti-system of the present invention is less important than a latex particle: determining the "agronomy" of the CRP. Detailed description of the present invention According to the present invention, a method for preparing IgY (AFc) from egg yolk is generally page 10 (please read the precautions on the back before filling this page) _0 · — Order P0! 587941 Intellectual Property Bureau of the Ministry of Economic Affairs Printed by employees' consumer cooperatives A7 B7 V. Description of invention (9 series include the following steps ... (1) Immune effect of spawning females; (2) Partial purification of yolk; (3) Immune affinity purification method; and ( 4) Extraction of antibodies. (1) Immunization of ovipositing females. Females of the geese, preferably ducks or geese, are immunized with an antigen to elicit one or more antibodies of interest. The antigen includes, but is not limited to, a microorganism (such as pathogenic or non-pathogenic bacteria, viruses, molds, protozoa, nematodes, etc.), a naturally occurring or synthetic protein (such as a toxin or Hormones), a naturally-occurring or synthetic accumulation, a recombinant protein or a fragment thereof, and any other substance capable of stimulating the production of antibodies, and combinations thereof. The antibodies can be expected from bird serum or birds It is obtained from the eggs. However, as mentioned earlier, the cost of collecting the antibody from the eggs is usually better in terms of cost. IgY and IgY (A Fe) are two isotypes. ) Transferred from the serum to the printed yellow. &Amp; Scientifically, the printed yellow of a duck egg contains about 4 mg of igY / ml and about 3-12 mg of gY (AFc), so each egg may provide 1 5-80 mg of I σ γ T w λ and 45-240 mg of I

IgY (A Fc). The volume of the printed yellow produced by # ～ 人 丄 A produced in any given time exceeds the volume of serum obtained from the birds by the female population. In addition, egg sound anti-limb stroke can be performed on a large scale without a large investment. The technique of immunizing a female with a selected antigen is well known, and is not limited to the use of a specific ", This technique uses any type of immune effect, which can be adapted to the volume. The present invention can be used, including subcutaneous, intradermal, muscular, and: routes to inoculate anti-office injections. Page 11 of this paper Standards are applicable to China National Standard (CNS) M specifications (2 丨 0 > &297; 297 meals (please read the precautions on the back before filling this page)

587941 A7 B7 V. Description of the invention (10) (Please read the notes on the back before filling this page) Preferably, a suitable adjuvant is co-administered with the antigen to enhance the immune response. In particular, a complete dose of Freund's adjuvant may be used alone or in combination with a subsequent dose of incomplete Freund's adjuvant. It has been found that the use of a suitable adjuvant is effective to maintain an antibody titer in the eggs of the immunized female for a long period of time, which makes it possible to efficiently produce the desired antibody. During the period of immunization, a female bird was initially inoculated with the antigen on day 0 and then received the antigen again after a period of time. The period of the initial immunization and the first booster administration and the period of the individual booster administration depend on the special characteristics of the antigen, and preferably at least two weeks. Usually within 10 weeks after the initial immunization, a large amount of reactive antibody system against the antigen is produced in the female body and the eggs it lays. The presence and titer of the special antibody against the antigen in the blood serum and eggs of the female can be confirmed by several conventional methods in this art. (2) Partial purification of egg yolks Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Partial purification procedures to remove most of the non-aqueous biomolecules and particles' and preferably remove most of the unrelated proteins in the egg yolks. Any conventional method that can achieve this can be used in the present invention. Examples include the use of PEG, dextran sulfate, or a natural viscose such as sodium alginate, carrageenan, and xanthan gum. The unwanted substance is co-precipitated, and an aqueous buffer or water is used to obtain an antibody-rich aqueous state substance. In a preferred embodiment of the present invention, first, the yolk and albumin on page 12 of this paper are again applicable to the Chinese National Standard (CNS) A4 specification (210X29 * 7 mm) 587941 A7 B7 V. Description of the invention (11 ) Separate and rinse with distilled water to remove as much egg white as possible. Pierce the vitelline membrane that covers the yolk, and you set the separated yolk separation part to dilute < with a large amount of aqueous buffer or water to form a suspension of yolk. The collected yolk is preferably diluted with an aqueous buffer or distilled water at a ratio of about 0 • 2 to about 1: 4 0 v / v, and Cheng Wenjia is 1: 5 to about 1: 3 0 v / v to dilute it. It has been reported that ψ 卬 PH is an important factor during the partial purification step (E · M Δ 1 '• Akita and S · Nakai, J. Food Sci. Vol. 57, p. 629 (1 993)). In terms of the best recovery rate, the pH is preferably set between the range of yolk antibodies printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. In this step, the temperature is preferably between 0 ° C and -60X: range. The yellow printed suspension was carefully stirred to form a homogeneous mixed Λσσ, and then allowed to stand for a time sufficient to form an aqueous and non-aqueous layer. And the% backward is to remove the water-insoluble microbe from the aqueous yolk suspension by centrifugation, which includes non-aqueous biomolecules, such as lipoproteins, lipid-filling, and solid vegetable dishes. The obtained antibody-containing supernatant can then be separated from the viscous precipitate by known methods such as pouring, extraction, and other methods. The yolk supernatant may optionally but preferably be further treated with non-denaturing salts at a concentration of _ ^ M-nan to induce precipitation of antibodies. Examples of acceptable salts for egg yolk antibody precipitation include, but are not limited to, τ, Na2S04, (NH4) 2S04, KCn, CaCl2, and MgS04. Preferred are Na2S04 and (NH4) 2S04. The concentration of salts used to precipitate the antibody is important and depends on the type of salt 'usually based on the final volume of the yellow ink supernatant in an amount greater than 15% by weight and less than 35% by weight' It is preferably in the range of 20/0 and 30% by weight. Page 13 This paper size applies to Chinese National Standard (CNS) Α4 size (210X 297 mm) (Please read the precautions on the back before filling this page)

1T 587941 A7 B7 V. Description of the invention (12) (3) Immunoaffinity purification method As used herein, the "immunoaffinity purification method" or, immunoaffinity chromatography, and the like terms refer to the antibody The type of separation based on the adsorption characteristics of a particular antigen. That is, the anti-system that binds to a particular antigen under a specific environment is separated from the unbound antibody under that environment. The present invention intends to use the immunoaffinity The use of purification methods to remove irrelevant proteins, including proteins other than immunoglobulins and non-antigen-bound immunoglobulins. More importantly, the procedure of the immunoaffinity purification method of the present invention unexpectedly completes one of the present invention The main purpose is to isolate the desired IgY (A Fc) antibody from all yolk antibodies including IgY. According to the present invention, the method of immunoaffinity purification is by applying an antigen immobilized to an insoluble carrier. The "antigen matrix" is composed. The type of the carrier is not important to the immunoaffinity purification method of the present invention. Any conventionally applicable A carrier substance that covalently binds to an antigen and does not respond to the interaction between the desired antibody and the antigen immobilized thereon can be used. The carrier is generally made of cross-linked agarose or tritium ~ Glucosamine made of ~ such as the CNBi * -activated agarose gel (Sepllarose) 4B which can be described from phar claw acia. The partially purified antibody system in step (2) is dissolved in a " The binding buffer is applied to the antigen matrix so that it forms an immune complex that fixes the antigen and the antibody to the egg n. Any buffer system that does not respond to the antigen-antibody interaction and can effectively maintain the desired binding conditions can be used in the present invention. The binding buffer is preferably selected from the group consisting of phosphate buffers. Page 14 This paper is _Family Standard (CNS) Α4ίϋ7Ικ) × 29 ^ 7 (Please read the precautions on the back before filling this page)-Order · Ministry of Economics Wisdom Printed by the Consumer Cooperative of the Property Bureau 587941 A7 B7 5. In the family consisting of the T3 solution, MES (2- [1 ^-? ^ Forest] ethanol luteol acid) buffer solution, and b 丨 s_τris buffer solution Among them, the MES buffer at the concentration of 20% is the best. As used herein, "flow-through substance," this term refers to an antibody solution flowing through the antigen matrix, which contains most of the non-binding substances. Based on general knowledge of immunoaffinity purification techniques, the ionic strength is higher than At 150 mM, the formation of an immune complex between about 7 and 9 is often recommended. However, under these conditions, egg yolk antibodies (especially the yolk antibodies from a duck yolk) seem to be less easy. In the best condition, it is combined with the antigen fixed on the chromatographic support. Active antibodies are still present in the flow-through substance. In the method of the present invention, it is used for γ (△ Fc) antibodies. The conditions of the immunoaffinity purification method are unique. In the present invention, the antibodies prepared by Inza are suitable for the environment of weak acid and low ionic strength, that is, the pH is in the range of 4-7 and less than 5 〇 under the ionic strength. The antibody is preferably in the range of pH 5-6 and more preferably in the range of 5.6_5.8, and interacts with the immobilized antigen. Under the binding conditions disclosed in the present invention , In the flow of matter, did not send Any detectable active antibody is available. (4) Extraction of antibodies The term "extraction solution" as used herein refers to the chemical solution A, D, and Ma that can separate the antibody bound to the antigen matrix. It contains an "extract" through the antigen matrix of the corpse. Generally speaking, the extraction conditions used to destroy the egg puertoplasma-antigen interactions are better than the destruction of mammalian antibody-antibody. Mei Liu ^ ^ Chou Yuanzhang's Impact of Interaction, page 15 (Please read the precautions on the back before filling out this page}, π Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs

587941 A 7 ~ B7 V. 'Explanation of invention (14) The conditions for raising are more moderate. (Please read the precautions on the back before filling out this page.) A typical eluent buffered at P below 4 or above 8 is suitable to eliminate IgY (A Fc) -antigen interaction. However, it is generally not recommended to use an eluent having an extreme pH value, because such a severe condition may cause a severe loss of the antibody's antigen-binding ability. In addition, the eluent containing a high concentration of a solubilizing agent can be used in the present invention. As used herein, "cha 〇tr 〇picagent" or "chaotrope" is a noun that can cause a change in the configuration of a protein molecule, such as the IgY (AFc) molecule of the present invention. Therefore, it is generally regarded as a protein denaturant. A solubilizing eluent interferes with the interaction between the antigen and the antibody by dissolving a hydrophobic binding region in the aqueous state. According to the present invention Most binding antibodies can be successfully eluted with any neutral buffer containing a mild concentration (> 1 M) of a solubilizing agent. In most cases, the solubilizing agent is removed after the elution. Will restore the natural protein structure.

The extracts used in immunoaffinity chromatography include, but are not limited to, pH 2.3 μM glycine-HC1, pH 10.0 μM glycine printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs. Acid-HC1, 6M of linidine-HC1, pH 3.0, 3.0M of potassium vaporization, 5M of potassium iodide, 3.5M of magnesium chloride, 3M of thiocyanamide / sodium thiocyanate, and 6 M of urea. However, for the activity of the recovered antibody, a mild ionic strength and a solubilizing neutral pH buffer, such as buffered in 20 mM MES buffer (pH 5.8) or 20 mM Tris (trihydroxyamidoamino) The 3 M sodium thiocyanate of (methane) (ρΗ 7 · 5) is preferably used in the practice of the present invention. The activation status of the collected antibodies can be easily printed by, for example, a low ion page 16 paper size applicable to China National Standard (CNS) A4 specifications (21〇297297 sauce) printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumer cooperatives 587941 A7 B7 V. Description of the invention (16) Yellow antibody to obtain an extract containing the IgY (A Fc) antibody. The printed yellow antibody system purified according to the method of the present invention is a homogeneous IgY (Z \ Fc). Its purity was checked by non-denatured sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular weight of the obtained IgY (AFc) was confirmed to be 120 kDa. No IgY contamination was found in the gel. Because of the multiple strain characteristics of the antibody, the isoelectric point of the IgY (A Fc) has a wide range (5.2-7.3). IgY (A Fc) purified according to the method of the present invention neither activates the complement system nor binds to rheumatoid factor in mammalian serum. The immunological reactivity between this IgY (A Fc) and mammalian IgG is not obvious. Therefore, the present invention also provides a new type of antibody suitable for clinical and academic research use. The present invention also provides various clinical and academic research uses of the IgY (A Fc) antibody prepared according to the present invention. For example, the present invention provides a method for immunizing an animal (including poultry, livestock, and pets) or a patient by administering to the patient a therapeutic amount of the IgY (A Fc) antibody of the present invention. To protect them from a variety of etiological substances including microbes such as bacteria, viruses, molds, protozoa, nematodes, etc .; and proteinaceous or non-proteinaceous substances such as allergens, toxins, Venom, hormones, or any other immunogen that can trigger an immune response. The purified IgY (A Fc) antibody is preferably administered in combination with a pharmaceutically acceptable carrier (such as water, physiological saline, etc.). The IgY (A Fc) antibody of the present invention can also be used to detect a human or animal I ---- 11imnr (Please read the precautions on the back before filling this page) Order

587941 Printed by the Consumer Property Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs A7 V. Invention Description (17) Among the body samples obtained from the objects, such as body fluids, tissues, and cell extracts of etiological substances of interest, including such as Non-pathogenic organisms, such as E. coli ~ / ζ ·, Salmonella enteritidis (heart / m⑽e // a to ... ..., and other bacterial organisms; a hormone such as estrogen, progesterone, sacral A major tissue-compatibility complex antigen, etc .; a tumor marker, such as α-fetal protein, prostate-specific antigen, etc .; a disease state marker, such as c-reactive protein, ferritin, etc. Use basis The lgY (A Fc) antibody obtained in the present invention can be a pathogenic substance of interest by any of the conventional methods well known in the art, such as single radioimmunodiffusion (SRID), immunoelectrophoresis, and radioimmunoassay (RIA), enzyme-labeled immunosorbent assay (ELISA), Western blotting method (WB), turbidimetric immunoassay (tia) or particle turbidimetric turbidimetric immunoassay to determine Or qualitatively detect it. Based on the optimal binding conditions defined above, the present invention further provides a quantitative or qualitative immunoassay method for analyzing a causative substance as described above, one of which is an antibody against the causative substance, preferably It is the purified IgY (A Fc) antibody of the present invention, which is cultured under the optimal binding conditions in the presence or absence of the causative substance. According to the present invention, the optimal condition is at a level below 50 mM. Under ionic strength and between pH 4_7. The antibody is preferably allowed to be in the range of pH 5_6, and more preferably is in the range of pH 5.6-5.8 to react with the causative substance. The invention also provides A kit for performing the above-mentioned immunoassay, wherein the kit includes an antibody specific for an etiological substance of interest. Formula η 彳, the antibody is preferably purified according to the present invention. ^ Fc ) Antibodies, page 19 This paper ruler is fine Chinese ^ standard (CNS) M rule woman ---------- »-----, order ------ · (Please read the first Note for this page, please fill in this page) 587941 Α7 Β7 V. Description of the invention (18) In the immunoassay method based on the ionic strength of lower than 50 mM and a pH of 4-7 is carried out at the conditions. The antibody is preferably in the range of pH 5-6, and more preferably in the range of 5 · 6-5 · 8 'to interact with the causative substance. When used in, for example, a single radioimmunodiffusion method (SRID), the IgY (A Fc) antibody of the present invention can be formulated by any conventional method in a method such as agar In a carrier medium composed of amine gel and the like. For example, a 'supporting medium' can be suspended in a buffer solution under heating, the IgY (A Fc) anti-system is added thereto, and the formed substances are mixed together. The resulting solution is poured into a glass plate or into a plastic container and then cooled to solidify. In order to supply the sample to be tested, a sample well is formed on the formed coagulation plate. Although the principle of this technique is well known to those skilled in the art, the physical requirements of the IgY (A Fc) antibody-antigen interaction are more stringent than the general physical requirements of antibody-antigen interaction. In particular, the duck's IgY (A Fc) resistance system only binds to the antigen under the specific binding conditions of the present invention and exhibits the properties of Shen Dian. The specific binding conditions can also be performed in other immunoassay methods (such as OchterlOly method (MO) and turbidimetric immunoassay (TIA)), where the primary antibody is caused by the IgY (A Fc ) The antibody-induced Shen Dian or agglutination system is considered a necessary step. Therefore, a preferred embodiment of the present invention provides a method for single radioimmunoassay analysis of an antigen, comprising: (a) preparing a salt concentration formulated at 1-50 mM and? 11 Agar gel of egg yolk antibody in buffer solution; page 20 This paper is a paper size standard (CNS) A4 specification (2 similar to 297 mm —) '-(Please read the precautions on the back first (Fill in this page) •• Qing · Order Printed by the Consumers ’Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 587941 A7 B7 V. Description of the Invention (19) (b) Digging several wells in 5 Xuanyang Mining 'and moving the father from the well', Ten meters of block; (c) Preparation of the dilution of the antigen in phosphate buffer solution physiological physiological difficulties; and-!.? (Please read the precautions on the back before filling out this page) (d) will each Dilute the antigen solution and sample into separate wells; (e) culture for at least 24 hours; and (f) measure the diameter of the immunoprecipitin circle, which is linearly related to the logarithm of the antigen concentration. In a quotient-labeled immunosorbent assay (ELISA) or particle-enhanced turbidimetric immunoassay, an antigen of interest placed in a sample is tested in a resin at an amount sufficient to interact with the sample, and is tested by immobilization on a resin. This result is captured by the IgY (A Fc) antibody on the plate. This result is the evidence of the antigen of interest. The resin test plate is such as polyvinyl chloride, polystyrene, etc., or a polystyrene latex, Fine particles made of polyester latex, polyvinyl chloride, bentonite, glass beads, etc. In the particle-enhanced turbidimetric immunoassay method, the captured antigen is then detected directly based on the change in turbidity. The change in turbidity was measured at 320-900 nm. In the tritium-labeled immunosorbent assay (E LIS A), the captured antigen was further detected by an antibody conjugated to a signal substance. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs for the implementation of the present invention. The following embodiments are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Page 21 This paper size applies Chinese National Standard (CNS) A4 specification (210X297 male sauce 587941 heart A7 each) V. Description of the invention (20) Implementation Example 1: Immune procedure for stimulating specific antibody production 1 2 only 16 weeks Dazhi's duck (Anas platyrhynchos va "domestica" for the production of antibodies and eggs of human arms) is separated on the Qin table. It accepts 1-5 mg / ml of C-reactive protein (CRp; 'Purified in sub-water] preliminary subcutaneous injection, the C-reactive protein formulated in a pH 7 $ 4 salt buffer was emulsified with an equal volume of $ Hachiman's adjuvant. The antigen used The concentration is generally in the range of Wang Fulun mg / ml. After this initial injection, the young one received four additional injections of i-5 mg of antigen five times in two weeks. Eggs were collected, labeled and stored under 4t until after beating, and taken and purified. During the test period, the extraction procedure of the additional knives was repeated every four weeks. Blood was performed on the 7th day after each additional injection. Π J. H. Miscellaneous blood samples and the resulting serum collected.-Example 2: Precipitated duck Physical requirements of the body In this example, the investigation of pH and salt on duck antibodies was jealous. The agarose powder was heated under the influence of suspended L ^ uz Μ 久 long silk and changes in sodium gas concentration Phosphate buffer solution. 1 Η Η Η, 5 亥 5 compound fat bran, 、, 右, and right are cooled to 56 ° C, at this time, 0.5 ml of the content is added. It is 0.4 mg / ml The purified CRP antigen solution was stirred. The solution was poured into a plastic container and cooled. The solids on the plate were made in the same room as "1". Antiserum well. The m-dipper made in Example 丨 was used to dilute the serum with the W buffer used in Yibei Rong plate, and the original 2 // 1 The antiserum and the diluted antiserum were placed in agarose flat pairs < separate wells. Page 22 iφφ ~ '{Please read the notes on the back ¾ before filling out this page] Order the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Standard for printing Zhongguanjia for secret use (-587941 A7 B7 V. Description of invention (21) In the first group of experiments, the Agarose gels with CRP were prepared at pH 4, 5, 6, 7, 8, and 9. The original antiserum and diluted antiserum 2/1 were placed in these agarose wells. After 48 hours of incubation, determine the diameter and shape of the immunoprecipitin circle, as listed in Table 1. Table 1 Diameter of the immunoprecipitin circle at different pH conditions. The antiserum is diluted in agarose. Buffer in the gel 1: 1 1: 2 1: 4 20 mM PB, pH 4.0---20 mM PB, pH 5.0 6.6 4 _9 3.9 20 mM PB, pH 6.0 6.3 4. .7 3.7 20 mM PB, pH 7.0 6.0 4, .5 3.5 20 mM PB, pH 8.0 5.9 4. .4 3.4 20 mM PB, pH 9.0 5.9 4,, 4 3.4 As shown in Table 1, no agarose plates at pH 4.0 were found Immunoprecipitin circle. Although the immunoprecipitin circle can be found in agarose plates with a pH greater than 5.0, the precipitin gradually becomes smaller in shape when the pH value is increased to 7. Based on the shape and size of the immunoprecipitin circle, the best precipitation effect can be obtained at pH 5.0. The results show that duck antibodies are suitable for binding to an antigen at p Η 5.0. Page 23 This paper size applies Chinese National Standard (CNS) Α4 specification (210X297 mm) (Please read the precautions on the back before filling out this page) ni ^ i— m 、 一 m ϋ · —ϋ · ϋ ml _ Economy Printed by the Consumer Cooperatives of the Ministry of Intellectual Property Bureau 587941 A7 B7 V. Description of Invention (22) In the second set of experiments, except at pH 5.0, each of the 20 mM contained 0.5, 1.0, or 1.5 M chlorine The agarose gel was prepared in sodium phosphate buffered saline, and the above procedure was repeated. After incubation, the immunoprecipitin circle was less clear than that shown on agarose plates without sodium chloride (data not shown). This result indicates that the precipitation of duck antibodies is significantly inhibited in the presence of salts. It can also be explained together that the agarose plate prepared in 20 mM phosphate buffer at pH 5.0 is more suitable for the formation of duck antibody precipitation. Example 3: Determination of the titer strength of the antibody The titer strength of the antiserum obtained in Example 1 was determined by SRID. The CRP-containing agarose plate was prepared using 20. mM phosphate buffer (pH 5.0) as described in Example 2. 2 // 1 The antisera obtained in Example 1 were each placed in a separate well dug in a flat plate. After incubation, an immunoprecipitin circle was formed around the well, and the circle diameter related to the potency of the antiserum inserted was determined. Example 4: Extraction of duck yolk-derived antibody The yolk system collected from the eggs laid by the highly immune duck in Example 1 was sufficiently washed with a weakly distilled water column to thereby Remove egg whites. The volume of the yolk was measured, and then it was thoroughly mixed with 10 times the measured volume of the yolk volume with distilled water. The mixture was then stored at .4 ° C for at least two hours, and then centrifuged at 10,000 with a Hitachi CR22F centrifuge on page 10,000. This paper size applies the Chinese National Standard (CNS) A4 size (210X297 mm) (please read the back first) Please note this page before filling in this page) mi m I ml, one C " Jn n ^ i ϋ— «in n Printed by the Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 587941 A7 B7 V. Description of the invention (23) (Please read the back first Note: Please fill in this page again) Centrifuge at rpm for one hour. A light supernatant layer and a semi-solid susceptible perturbation layer were formed in the centrifuge tube. Carefully collect the supernatant layer, add powdery ammonia sulfate to it and stir it slowly until the final concentration is 25 g of ammonia sulfate per 100 ml of egg yolk extract, so that the egg yolk antibody can be used. It is completely salted out. The Shen Dian system is due to 4. Centrifuge for 30 minutes at 10,000 rpm and collect. After decanting the supernatant, the obtained pellets were re-dissolved in a suitable buffer solution and dialyzed with the slow hydration solution to remove the excess ammonia sulfate. Twenty batches of the crude antibodies obtained by this procedure were aggregated and stored at 4 ° C 'for further experiments. The titer strength of the aggregated antibody was determined by the SRID analysis method described in Example 3, and the diameter of the immunoprecipitin circle was 6.1 mm. Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Example 5: Covalent attachment of an antigen to cyanogen bromide-activated agarose matrix A CRP solution was prepared in a 0.1 M carbonate buffer, pH 8.5, And the concentration is 5 mg / ml. The cyanogen bromide-activated agarose gel 4B from Pharmacia was first washed with HC1 at 10 mM ice in an amount that is 10 times the volume of the matrix, and then allowed to react with the matrix volume at 4 ° C. Two times the amount of CRP solution was reacted overnight. Coupling efficiency is calculated by dividing the amount of coupled protein (in units of A2 80) by the amount of the initial protein (A2 80). The results show that the coupling efficiency of the CNB-activated agarose gel (S e p a ar s s e) 4B falls within the range of 80-85%. For later use, the antigen matrix was suspended at 4 ° C in a ratio of 1: 1 (v / v) to 0.5 mM ethanolamine in 20 mM Tris-HCl (pH 8.5). ) 2 hours to block the action of the protein that still exists. Page 25 This paper applies Chinese National Standard (CNS) A4 (210X297 mm) 587941 Μ B7. 5. Description of the invention (24). The antigen matrix was then washed with PBS containing 0.02% sodium azide and stored at 4 ° C. Example 6: Immunoaffinity Purification of Egg Yolk Antibodies In the following Examples 6-8, the duck antibody obtained from Example 4 and the antigen matrix system prepared in Example 5 were used to illustrate the use of this method for egg yolk. Suitable binding conditions for antibody immunoaffinity purification. 1 ml of the antigen matrix is packed in a common column and immersed in one of the binding buffers listed in Table 2. The antigen matrix was allowed to react with 0.25 ml of antibody in the same binding buffer. The antigen matrix was washed with the binding buffer until the effluent was substantially free of protein. The bound antibodies were subsequently extracted with 4M quinidine-HC1, and after complete dialysis, their optical density was measured at 280 nm. The binding capacity of duck antibodies in different binding buffers is expressed in terms of the amount of antibody in the final extract, as shown in Table 2 below. 0 (Please read the precautions on the back before filling (This page) ——Order ------ Employees' Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs prints the second volume of physical resistance. Zhongwu Tichonghai-5 Page 6 2 The size of this paper applies to Chinese National Standard (CNS) A4 (210X297mm) 587941 A7 B7 V. Description of the invention (25) 丨 ^ Combined with buffer solution (A 2 8 〇 unit X m 1) 20 mM PB, pH 4 0 • 26 20 mM PB, pH 5 0,, 43 20 mM PB, pH 60, .39 20 mM PB, pH 7 0 ·, 32 20 mM PB, pH 8 0_, 29 20 mM PB, pH 9 0., 25 A considerable amount of duck resistance system was found to be bound in a weak acid environment. Example 7 The procedure of Example 6 was repeated except that the pH value of the binding buffer of each test group was adjusted to 5.2, 5 · 4, 5.6, or 5.8. The results are shown in Table 3 below. (Please read the notes on the back before filling this page)

Order% ·. The Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs prints the physical resistance of Table 3, the upper part of the bottom line is the same as the original, and it is not resistant to the combined physical resistance. This paper scale applies Chinese National Standard (CNS) A4 specification (210X297 mm) 587941 A7 B7 V. Description of the invention (26) Binding buffer (A28q unit xml) 20 mM PB, pH 5.2 0.45 20 mM PB, pH 5.4 0.46 20 mM PB, pH 5.6 0.53 20 mM PB, pH 5.8 0.54 In Table 3, the environmental system at pH 5. 6-5. 8 shows that it is most suitable for the binding of this duck antibody. In Example 8, the procedure of Example 7 was repeated at pH 5.8 except that the phosphate-binding buffer was replaced with a MES buffer or a Bis-Tris buffer. The results are shown in Table 4 below. Brother 4 shows the amount of antibody bound to the antigen matrix under different conditions. The amount of antibody in the extract is bound to the buffer (A28〇 unit xml) 20 mM PB, pH 5.8 0.54 20 mM MES, pH 5.8 0.68 20 mM Bis-Tri s, pH 5.8 0.63 (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs This result illustrates the 20 mM MES buffer solution adjusted to pH 5.8 The most suitable binding buffer for immunoaffinity purification of duck antibodies. Page 28 This paper size applies the Chinese National Standard (CNS) A4 specification (210 × 297 mm) 587941 A7 B7 V. Description of the invention (27) Example 9: In addition to the use of 1-3 Μ in the extraction solution The procedure of Example 6 was repeated by using 20 mM MES buffer as the binding buffer (pH 5.8) with sodium thiocyanate (pH 7.5) as a solubilizer to compare with 4 M of quinidine-HC1. The extraction efficiency of the 4 μm quinidine-HC1 was functionally defined as 100% as a reference standard. The relative efficiencies of other eluents are determined by dividing the unit of A280 which is extracted by each of the eluents by the unit of A280 which is 4M of tarsalite-HC1. The results are shown in Table 5 below. (Please read the precautions on the back before filling in this page) Table 5 Efficiency of Eluent Solution Efficacy of Extraction Solution 4 mM Trinidine-HC1, pH 8.0 100 3 Μ Sodium Thiocyanate, p H 7.5 95 2 Μ Sodium thiocyanate, p H 7.5 70 1 Μ Sodium thiocyanate, ρ H 7.5 55 ---- Ordered by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs, printed in Table 5, it can be seen that the 3 MH thiocyanate Sodium, which elutes about 95% of the bound antibodies from the antigen matrix, is almost as efficient as 4M of quinidine-HC1. Example 10: Purity and activity of duck antibodies on antigen substrates purified by immunoaffinity. Page 29 This paper applies Chinese National Standard (CNS) A4 specification (210 × 297 mm). 587941 Employees of Intellectual Property Bureau, Ministry of Economic Affairs Printed by the cooperative A7 B7 V. Description of the invention (28)

In order to detect the composition of the antibody produced according to the present invention, analytical SDS-PAGE was performed on an 8% non-reducing polypropylene ammonium gel, to which 60 # g of the crude antibody and 40% were added. § The flow-through substances collected in Example 3 (the second and third runways respectively), and 20 " g of 3 M sodium thiocyanate (pH 5 · in Example 9) were added. 8) The extracted antibody product (runway 4). The results are shown in Figure 2. The crude product and the flow-through substance both show two distinct 180 kDa and 120 kDa bands (these sizes are measured by molecular weight markers in the second runway), which are The IgY and IgY (Z \ Fc) relative mobility of the duck were respectively matched. On the other hand, the affinity-purified anti-system was composed of an IgY (A Fc) antibody, which showed a single band on the gel. These results prove that the method of the present invention is an excellent strategy, and the desired IgY (A Fc) can be separated from the aggregation of the immunoglobulin mixture composed of both IgY and IgY (AFc). )antibody. Almost all IgY antibodies do not bind to the antigen matrix and are present in the flow-through substance. Density analysis of the gel showed that more than 95% of the purified antibodies were homogeneous IgY (AFC). In addition, no more than 60% of the total protein of the crude antibody is IgY (AFC) (data not shown), and 24% of the total protein of the crude antibody (which is believed to be equivalent to the The desired amount of IgY (AFc) can be bound to the antigen matrix in an affinity purification step. The activity of the purified antibody was determined by SRH) as described in Example 3. The diameter of the purified Shen Diansu circle of the purified antibody is page 30. The size of this paper is applicable to China National Standard (CNS) A4 ((Please read the precautions on the back before filling in this page)

587941 V. Description of the invention (29), which is only slightly smaller than the crude antibody 6.1 Duck anti-CRP antibody (please read the precautions on the back before filling this page) Example 11: Determine the CRP concentration by the SRID method Except for the examples Substitute the affinity purification in 10 to replace CRP and add to the lipid-free gum plate to a final concentration of: 2xb, as described in Example 2 using 20㈣ dish solution (PH 5.0). The system borrows Wang Fuzhi gum tablet. Dose of human serum from a cRp-positive provider was added to a separate well dug in a fixed interval on a plate, and cultured for 48 hours. The straight line of the immune Shen Diansu circle was measured and plotted against the logarithm of the concentration of the CRP standard to obtain a standard curve. Over the entire measurement range: Department, ... as shown in the figure. CRp concentration in unknown samples; estimated by interpolation of the calibration curve of the two calibrations. The result obtained by the SRID method in the present invention is completely the same as that obtained by Denka Seiken purchased from Tokyo, Japan.

Co., Ltd. '3-4-2 Nihonbashi Kayabacho, Chuo-ku ’s commercial CRP-latex kits are consistent with the results (Figure 4 Example 12: Determination of CRP concentration by ELISA method Ministry of Economic Affairs Intellectual Property Bureau Consumer Consumption Cooperative printed the duck anti-CRP anti-system obtained by affinity purification in Example 10 with a coating buffer (coat ngbuffe Γ) (20 m μ salt-filling buffer, ρΗ 8 · 2) Dilute to a final concentration of 16 / ig / ml. Then, the obtained solution of 100 // 1 is dispensed into each well of an ELISA plate (purchased from Costar Co., Ltd.). The plate was cultured overnight at 4 ° C or 3 hours at 37 ° C. At the end of the cultivation, 200 // 1 was added to each well at page 31. This paper size applies Chinese National Standard (CNS) A4 specifications ( 210X297 mm) 587941 A7 ______ B7 5. Blocking agent (30%) of skim milk powder formulated in coating buffer solution (30%) to stop IgY (A Fc) from coating on the plate. Continuously diluted crP was prepared at room temperature with 4 in a reactive buffer (MOmM in 20mM Tri). The coated duck antibody in NaCl) buffer solution interacted for 30 minutes, and then the plate was thoroughly washed with a washing buffer (reactive buffer containing 0.05% tween_2). A dose of goat-anti-CRP antibody (purchased from GOOd Biotech Corp) diluted in reactive buffer was added to each well ', and the plate was cultured for 30 minutes, and then washed with the The buffer was washed thoroughly. Next, a dose of HRP-conjugated rabbit-anti-goat IgG (Sigma) diluted in the reactive buffer was added to each well, and the plate was incubated at room temperature for 30 minutes. Then it was thoroughly washed with the washing buffer solution. 100 // 1-0-Signiamine was added to each well 'as the substrate of the conjugated enzyme. The plate was incubated at room temperature for 15 minutes. The reaction was neutralized with 1 00 // 1 of 1N H2S04. The absorption value of each well at 4 5 Onm was measured and plotted in Figure 5. This test Sensitivity can be as high as 2.5 ng / ml (OD > 1). R-squared can be as high as 0.9941. Example 13: τιA formula with particle enhancement (LAT: Latex Immunoturbidimetric Analysis Method) Determine the concentration of CRP. The dream step of coupling anti-CRP IgY (△ Fc) to the latex particles is at 4 ° C, at a concentration of 100% # 100 (w / v) latex suspension, 400 // 1 of 20 mM Tris buffer (ρΗ7.2), 10 mg / ml of duck anti-CRP antibody purified from the actual batch of Example 10, and 0.1% of The inverse tθI of sodium azide should be performed in the mixture for 2 hours. 5 ml contains 1% bovine blood μ @ 丨! 2: G lu: | Γ: ---------------- 11 ------ (Please read the precautions on the back before filling this page): _ Intellectual Property of the Ministry of Economic Affairs Printed by the Bureau's Consumer Cooperatives 第 32 页

This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) 587941 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs V. Invention Description (31) (BSA; purchased from Sigma) 20 mM Tris buffer (PH7.2) was added to the latex-IgY (ΔFc) mixture, and the obtained suspension was cultured at 4 ° C for 1.5 hours, and stirred. After centrifugation at 15,000 rpm for 1 hour at 4 ° C, the collected pellets were resuspended in 5 ml of 20 mM Tr1S buffer (PH7.2) containing 1% BSA., For subsequent use. The CRP-containing calibrator sera was used as a standard antigen, with a high degree of: 0, 1, 5, 10, and 20 nig / di, and a low range of concentrations: 0, 0.5, 1, 1.5, and 21 ^ / (11. The two additives are R1: 20 mM Tris buffer (pH 7.2) and R2: IgY (△ Fc) -hybrid latex suspension suspended in R1. The cause The increase in the absorption value (at 5 70 nm) caused by the mixing of R2 with the standard antigen was measured with the turbidity automatic analyzer of Hitachi 7020 at the corresponding time, and the data obtained are for the low range and high range respectively. The CRP concentration is plotted as shown in Figures 6a and 6b. Therefore, the CRP concentration in the unknown sample can be estimated by interpolation of the standard curve. All patents cited in the specification of the present invention And literature reference materials are fully incorporated here as reference materials. If there is any conflict with this case, the description (including definitions) of the present invention shall prevail. At the same time, the present invention has also been made using the above-mentioned special embodiments. Explain that it must be understood that 'is obvious to those skilled in this skill Various improvements and changes can be made without departing from the spirit and scope of the present invention. Page 33 This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) LT—! (Please read the first (Please fill in this page again for attention) Η

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Claims (1)

587941 A method for preparing an antibody IgY (AFc) from egg yolk, the antibody system does not substantially have an Fc-equivalent region, the method comprises the following steps: (a) immunizing a female poultry with a selected antigen, So that the poultry antibodies raised by the antigen can accumulate in the yolk; (b) collect the yolks of the immune females, and remove the non-aqueous biomolecules and particles, so as to obtain a water-soluble yolk-containing antibody (C) Passing the distantly water-separating separation portion through an inert support matrix having the antigen immobilized thereon at an ionic strength of less than 50 mM in the range of pH 4-7. The formation of an immune complex of the immobilized antigen and the yolk antibody; and (d) separating and extracting the yolk antibody from the immobilized antigen. 2. The method according to item 1 of the scope of patent application, wherein in step (b), the collected yolk is diluted with a large amount of aqueous buffer solution or water, and then centrifuged or filtered to remove the non- Water-based biomolecules and particles. 3. The method according to item 2 of the patent application range, wherein the collected yolk is diluted with an aqueous buffer solution or water of about 1: 5 to about 1: 3 0 v / v. 4 · The method according to item 1 of the scope of patent application, wherein in step (b), the non-aqueous biomolecules and particles are repeatedly degassed by PEG, dextran sulfate, or a natural viscose * and then centrifuged Or remove it by filtering. 5. The method according to item 1 of the scope of patent application, wherein before step (c), the yolk resistance system in the water-soluble portion is further selected from the paper standard to apply China National Standards (CNS) A4 specifications ( 210 X 297 mm) " ------- A8 B8 C8 The patent application Fanyuan D8 KC1, CaCl aii2 and then redissolved in NaCl, Na2S04, (NH4) 2S04, KC1, MgS04 non-denatured salts and precipitated solution. For example, the method of claiming the scope of the patent claims, wherein in step (e), the immune complex is formed at a pH in the range of 5-6. 7. The method according to item 6 of the patent application range, wherein in step ⑷, the immune complex is formed at a pH ranging from 5.6 to 5.8. 8. The method according to item 丨 in the scope of patent application, wherein in step (0), the immune complex is selected from the group consisting of a phosphate buffer solution, mes (2_ [n_morphine] ethanolsulfonic acid) buffer solution, And a bis-Tris buffer composed of a buffer of the population. 9 · The method according to item 8 of the patent application, wherein the immune complex is formed in 20 mM MES buffer. 10 · If the patent is applied for The method of the item i in the range, wherein in the step; step (^), the yolk antibody is separated from the antigen by borrowing a solubilized salt or at a pH lower than 4 or higher than pH 8. u. If applied The method of item 1G of the patent, wherein the method of item 1 of the patent scope is as described above, wherein the method is selected from the group consisting of 4 arboridine HC1 and ^ 3M sodium thiocyanate. 12. ^ Apply for a patent 帛According to item 丨, the female poultry line to be immunized is a black-eyed pterodactyl. 13. For the method of applying for patent No. 12, the female poultry line is as described in item No. 1 of the patent scope. Method, wherein the inert carrier matrix is -2- 15. 16. 17. by cross-linked agarose or According to the method of applying for patent No. 丨, the antigen is selected from the group consisting of voronogenic or non-pathogenic microorganisms, naturally occurring or synthetic oligomeric group proteins or The fragment and the combination thereof. The method of claim 1 of the patent scope, wherein the female poultry is immunized with an antigen conjugated with a water-in-oil emulsification adjuvant. A method for preparing an antibody IgY (AFC) from Immediately, the antibody system basically does not contain an Fc-equivalent region, the method includes the following steps: U) immunize a female poultry with a selected antigen So that poultry antibodies raised by the antigen can accumulate in the yolk; (b) dilute the immune with an effective amount of water because p Η is about 5-7 and the temperature is about 0 ° -6 ° C. The yolks of the females are collected for at least one hour to form a mixture to collect the yolk antibodies; and (c) by centrifugation at a rate of 1,500-30,000 rpm for about 0.5 at 0 ° C-60 ° C -6 hours, or filtered through filter paper, The water-soluble separation portion containing the yolk antibody was separated from the granules and lipids; (d) The yolk antibody in the water-soluble separation portion 8 was precipitated with amine sulfate or sodium sulfate; (e) in a salt of 1-50mM At a concentration and a pH of about 4-9, the egg yolk antibody is re-dissolved with a buffer solution; and this paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 587941 8 8 8 8 AB c D范围 Application scope (f) Add the solubilized egg yolk antibody to a chromatographic carrier substrate on which the antigen is immobilized to immunoadsorb the egg yolk antibody; (g) Wash the chromatographic support matrix with a buffer solution at a salt concentration of about 4 to 9; (h) Elute with a pro-solvent at a concentration higher than 50 mM or a buffer with a pH lower than 4 or higher than 8. The yolk antibody bound to the chromatographic support matrix is obtained to obtain an extract containing the IgY (AFc) antibody. This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)