TW587941B - Manufacture of IgY(DeltaFc) antibodies and uses thereof - Google Patents
Manufacture of IgY(DeltaFc) antibodies and uses thereof Download PDFInfo
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587941 A7 ________ B7 五、發明説明(1 ) 發明之背景 1) 本發明之範疇 (請先閲讀背面之注意事項再填寫本頁) 本發明係有關於一種自鳥類印黃製備及純化IgY(A Fc) 抗體的方法,以及藉此方法所產生之IgY(A Fc)抗體。本 發明亦有關於將該新穎之IgY(A Fc)抗體用於定量或定性 分析一感興趣之病因抗原。 2) 相關技藝之說明 抗體係已廣泛使用於多種生物研究及臨床的應用。由 高度免疫性之哺乳動物所獲得之血清係為多株抗體最常見 之來源。由此等免疫血清所得之抗體係屬於一般所稱之 “免疫球蛋白’’之蛋白質的族群中,於該免疫球蛋白中免 疫球蛋白G (I g G)是最多的。該I g 〇分子係由三個區域 (domains)所組成,其中有二個Fab區域及一個Fc區域。該Fab 部位主要涉及抗原的結合。雖然該Fc部位不具有與抗原結 合的能力,但其係管理一抗體的多種生物活性,諸如補體 結合(complement fixing)及Fc受體的結合。 經濟部智慧財產局員工消費合作社印製 於免疫診斷學之技術中,一完整的I g G分子並不適用於 涉及哺乳動物jk清之偵測系統及免疫分析之中,其係因為 於IgG分子上的Fc區域係具有與Fc受體結合、活化該補體系 統、以及與哺乳動物血清中之類風濕因子(rheumatoid factor)起反應等能力。IgG分子之Fc部位的移除一般常導致 此種干擾的降低(E. Lamoyi等人於1986年發表於“酵素學 方法(Methods in Enzymology)” 、第 121 卷、652-663 頁)。 第3頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) 587941 A7 B7 五、發明説明(2 ) 於免疫治療中之抗體的某些建議使用係包括治療患有 毒殺性之細菌毒素或蛇毒之病患(如美國專利案號第 5,340,923及5,601,823號中),以及剛出生之小豬對致命腸道 大腸桿菌病的預防(H. Brussow等人.,j. ciin. Microbiol. 第 25 卷、982 頁( 1 987);以及 C.〇.Tacket 等人·,New Eng. J.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.第318卷、1240頁( 1 988))。已知一抗體分子之Fc區段 係為該免疫球蛋白最具抗原性之部位(Ε· M. Akita等人 J Immunol. Methods.第 162卷、1 55- 1 64 頁( 1 993)),貝1J J:匕區段 之切除(其會造成該F (a b ’)2區段的形成)將明顯降低多數位 於免疫球蛋白分子上之潛在的過敏原位置,因此對施用該 免疫球蛋白之人類或動物是有益的。 最近已顯示該雙鍵結之F(ab’h抗體區段係較適用於該 免疫診斷測試中(M· Mumsugu 等人,j. c〇ilc)ld Intei.faee Sq 第⑷卷、378 頁(1991);以及 j. L.〇rtega_Vinuesa# 人,J· Immunol Methods 第 90卷、第 29 頁(1996)),且比 本身更適用於涉及哺乳動物血清之免疫分析的發展中。 然而’該F(ab,h抗體區段並未如一般人所希望之廣泛 使用於臨床免疫診斷套組中。其可能歸因於該f(礼屮區段 經濟部智慧財產局員工消費合作社印製 (請先閲讀背面之注意事項再填寫本頁) Φ0! 之大規模生產的困難度與不符合經濟效率之原因,节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
F(ab’)2區段於傳統上係藉由IgG經胃蛋白 沾A 史曰的水解,而後經 層析法純化而製得。 鴨子與其等分類學上關係相近者 可M及σ卩分爬蟲類(諸 如龜)係具有三種血清免疫球蛋白:一 £刀子免疫球蛋白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 (鴨子中為800 kDa),以及各二種沈降係數為'Μ (鴨子 第4頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210Χ297公羡) 587941 A7 ______ B7 五、發明説明(3 ) 中為180 kDa)與5.7S (鴨子中為130 kDa)之低分子量];gG的 異構形式(E. R. Unanue 等人,J· Exp. Med.第 121 卷、 (請先閲讀背面之注意事項再填寫本頁} 697-714 頁( 1965); Η· M. Grey,J. Immunol 第 98 卷、811-819IgM (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
頁 (1967);以及B. Zimmerman等人·,生物化學 (Biochemistry)第 10卷、482-44 8 頁(197 1))。因為鳥類之 IgG 係存在於卵百中’故一般稱為I g γ。該由較短之重鏈所構成 之5.7S的IgY在結構及抗原性上相似於該7.8S IgY之F(ab,)2 區段(如第1圖所示),此事實導致利用IgY (相當於7.8S IgY) 及IgY(AFc)(相當於5.7S IgY)之命名來表示二種IgY之異 構形式(Κ· E. Magor 等人,j. immun〇i·第 149 卷、 2627-2633頁(1992))。 經濟部智慧財產局員工消費合作社印製 於感染或實驗性免疫化鳥類中所進行的研究顯示該鴨 抗體係缺乏多數生物效應功能,其包括補體的結合以及Fc 受體之結合,但並未犧牲其等對於對應抗原的結合活性(G. W. Litman 寻人·,Immunochemistry 第 10卷、323 頁(1973); 以及 Τ· E. Toth 等人,Avian Dis·第 25 卷、17-28 頁 (1981))。其可能起因於該ι2γ(Δ Fc)抗體之FC-對等區域的明 顯缺無’該I g Y (△ F c)抗體係構成該鴨抗體反應的主要部 分。因而相信,假若可發現一值得信賴之製造該抗體的方 法’且可鑑定出適於其活性之物理性要件,則該與F(ab,)2 區段在構造及功能上相似之IgY(A FC)抗體將可於免疫應 用上提供不凡的益處。 經報導,鳥類卵黃抗體係如哺乳動物之抗體一樣,存 在有對研究及臨床應用上有用之特性。(如美國專利號第 第5頁 本紙張尺度適用中國國家標準(CNS ) M規格(2丨0><297公釐) 587941 A7 B7 五、發明説明(4 ) 5,340,923; U.S. 5,5 85,098; U.S. 5,60 1,823;及 U:S. 5,976,5 1 9)。由一產卵雌禽所得之卵黃係比高度免疫性哺乳 動物血清更便宜且操作上更方便且更安全。更重要的是, _黃抗體可順應於現代動物保護的相關法律規範(A. Poison 等人,Immunol. Commun.第 9卷、475 頁( 1 980);以及 B· Gottstein等人)。此等事實暗示卵黃有潛力使用作為一 抗體的商業來源。 現已致力於自卵黃分離及純化IgY。例如包括洋菜、黏 膠質(於1989年2月8日所公開之日本公開案第64-38098號 中)、硫酸葡聚糖(J. C. Jensenius 等人,J. Immunol. Methods 第 46卷、63 頁(1 98 1))、天然樹膠(H. Hatta 等人, 食品科學雜諸(J· Food Science)第53卷、425頁(1988))及聚 乙二醇(PEG) (A. Poison 等人,Immunol. Invest·.第 14 卷、 323頁( 1 985);亦可見於頒予A. Poison之美國專利第 4,550,019號中)等物質係被用於沈澱非水性之生物分子,主 要係為脂質及卵黃顆粒,以藉此收集一含有豐富卵黃抗體 之水可溶相。A. H a s s 1等人發展一種兩步驟之層析方法,其 包έ疏水父互作用層析法以及尺寸區別層析法,以進一步 自一經PEG-純化的分離部分中分離出該卵黃抗體(a. Hassl 及 H Aspock, J. Immunol. Methods 第 11〇 卷、225 頁 ( 1 988))。Akita等人已描述一種用於分離igY之改良的方 法’其中卵黃抗體係藉由以大量體積的水稀釋該卵黃,而 後將所獲得之上清液進行尺寸區別層析法以及/或離子交 換層析法,而自雞蛋中萃取而得(E. M. Akita等人,J. 第6頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210XΜ?公釐) (請先閱讀背面之注意事項再填寫本頁)(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)
訂 經濟部智慧財產局員工消費合作社印製 587941 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明説明(5 )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·第 160 卷、207 頁( 1 993);以及 Ε· M. Akita 與 S· Nakai,J. Food Sci.第 57卷、629 頁( 1 993))。 然而’此等所有的研究及專利皆僅集中注意於從鳥類 卵黃中分離出全體卵黃抗體(其包括至少IgY及IgY( △ Fc)),而非專注於單獨igY(A Fc)抗體的分離。再者,因該 IgY(AFc)抗體係僅存在於雁形目(〇rcjer Anseriformes)之鳥 類(諸如鴨及鵝)中,故於雞形目(galllfom birds)(諸如雞及 火雞)上進行之分離方法無法令人聯想到一 IgY(A Fc)抗體 之成功純化。 於1 989年中,Higgins嘗試自高度免疫性之鴨血清中製 備該等抗體’但該於pH 8.0及0.5 M NaCl下親和純化之抗體 大致上無法發揮有效的沈澱反應或凝聚反應(D. Α· Hlggins, Comp. Biochem. Physiol.第 93B 卷、135-144 頁( 1.989))。如 Hlggins於文獻中所聲稱者,用於形成鴨抗體沈澱素之最佳 pH值係在pH 8.5至pH 9.05之範圍間。而後,沒有任何有關 於IgY(A Fc)抗體之分離及其等可能之使用的重要研究被 發表。 因此,於本技藝中存有一個對於快速、低成本且高效 盈之方法的需求’該方法可自全體抗體中簡易分離所欲之 IgY(AFc)抗體,同時可維持其等之活性。再者,於本技藝 中係有一個對於經實質純化之IgY(AFc)的需求,其可作為 一種新類型的F(ab,h抗體,以供各種免疫診斷及免疫治療 之用。 本發明之概述 第7頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210x297公酱)----- (請先閱讀背面之注意事項再填寫本頁)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 、發明説明(6 ) 吾人已進行廣泛的研究,以滿足上成、廿 業需求。驚人的是,我們發現一 ^1卵5抗體之產 "、目鳥類你龙 抗體之成功的分離方法,其俜 汽之1gY(A Fc) 驟期間,在該抗原及抗體間交互作用曰\”和性純化步 透過一簡單的程序而輕易地達成。取仏結合條件下, 由本日日+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 +
於經濟效益下,簡單地生產出具有一 ^月之方法,可 F(ab,)2抗體,例如該已純化之。以厶產辜之新類型的 Fc)抗體係可使用於各種免疫之應用上且所產生之IgY(A 因此,本發明之一目的係欲提供一 蛋中製備IgY(A Fc)抗體的方法。該方法一於自雌禽所生之 驟:以一免疫原免疫該家禽之滩食//λ奴包含下列之步 #宵、從雌金& 分純化出全體抗體、以及對抗該 史中# ^ ^ t 兄反原所生之抗體的免疫 親和性純化,其中於免疫親和性 、、七化步驟中,該抗體與免 疫原/抗原的結合係於弱酸及低離子強度之環境下進行。特 別的是,該抗體-抗原的交互作用係於一低於5〇 mM之離子 強度下’在pH 4-7的範圍内進行,以獲得一最佳的結果。 因此,本發明係提供一用於自卵黃製備IgY(A Fc)的方 法,其包含下列之步驟: (a) 以一選定抗原來免疫化一雌性家禽,以使得該抗原 所引發之豕禽抗體可累積於印黃中; (b) 收集該免疫化雌禽的卵黃,且移除其内之养水性生 物分子及顆粒,以藉此得到一包含卵黃抗體之水溶 性分離部份; (c) 於一低於50 mM之離子強度下,在pH 4-7的範圍 第8頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210><297公董) (請先閱讀背面之注意事項再填寫本頁)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)
-訂- 經濟部智慧財產局員工消費合作社印製 587941-Order- Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 587941
五、發明説明( 内’令該水溶性分離部份通過一其上固定有該抗原 的惰性擔體基質’以允許該固定抗原與該卵黃抗體 之免疫複合物的形成;以及 (d)自该固定抗原中分離及沖提出該卵黃抗體。 本發明之另一目的係在提供一由本發明之方法所製造 之新穎的IgY(A Fc)抗體。本發明之又一目的係將該所產 生之IgY(A Fc)抗體提供於臨床及研究上的應用。除了低 成本及製備容易外’本發明之IgY(A ?勾抗體係具有不活 化哺礼動物血清中之補體系統及類風濕因子之優點,同時 對哺乳動物之IgG具有不良之交互反應,因此,特別適用 於涉及哺乳動物之血清的免疫分析,且具有最小之干擾。 如熟習該項技藝者所知,該抗體可以單一試劑之方式存在 而用於臨床、學術研究、以及其他的應用上,或者可包含 於一商業化之套組中,以作為一活性成分之方式存在。 本發明之另一特殊目的係欲提供一用於一感興趣之病 因物質之免疫分析的試劑,其包含一由本發明之方法所製 得之IgY(A Fc)抗體。 本發明之又一目的係欲提供一免疫分析之方法以及一 用於進行該免疫分析之商業化套組,其中一對抗一病因物 質之抗體(較佳係為由本發明所純化之IgY(A Fc)抗體)係 於一結合至該病因物質之最適條件下,於該感興趣之病因 物質存在或不存在之下培養,以藉此定量或定性分析該病 因物質。依據本發明,該最適條件係為於一低於5 0 m Μ之 離子強度下,pH在4-7範圍内的條件。 ' 第9頁 本紙張尺度適用中國國家標準(CNS) Μ規格(2丨0><297公羞) (請先閱讀背面之注意事項再填寫本頁)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 ««: 經濟部智慧財產局員工消費合作社印製 587941 五、發明説明( 經濟部智慧財產局員工消費合作社印製 圖式之簡要說明 本發明之上述及其他目的與特徵將 i 體實施例及所伴隨之圖式的敘述而變得更明歹1之較佳具 JUJU 人乃白,立φ 弟1圖係為一顯示典型之靈長類動 /、 異構形式的示意圖; 的1扣與鴨子IgY 第2圖係顯示於本發明中所製備之二 SDS-聚丙烯醯胺凝膠電泳進行之狀況, '、抗於 (Co⑽assie Blue)染色之狀況: 1以科麥西藍 中;該粗萃之抗體萃出物於第二跑道中第一跑道 免疫親和性層析法之流經物質於第三跑道中·=抗體通過 物中之親和純化抗體係於第四跑道中;’以及於沖提 第3圖係顯示C·反應性蛋白質(⑽之 沈澱圈之直徑對應於血清桿準σ ·、.果免反 Θ铩羊σα之反應性 (mg/dl,取對數等級)作圖; 变曰貝派度 第4圖係顯示-依據本發明膽分析法以及—藉一商 業化之免疫比濁分析所獲得之CRp濃度的比較· 第5圖係說明一ELISA分析,其中本發明之㈣△&)抗 體係用於決定該CRP的濃度,且 第6a-b圖係說明一顆粒增進性比濁免疫分析其中本 發明之祕Fc)抗體係與一乳膠粒子相輕二用:決定 該CRP的》農度。 本發明之詳細說明 依據本發明,一用於自卵黃製備IgY(AFc)之方法一般 第10頁 (請先閱讀背面之注意事項再填寫本頁) _0·— 訂 P0! 587941 經濟部智慧財產局員工消費合作社印製 A7 B7 五、發明説明(9 係包含下列之步驟··( 1 )產卵雌禽的免疫作用;(2)卵黃的 部分純化;(3)免疫親和性純化法;以及(4)抗體的沖提。 (1)產卵雌禽的免疫作用 一雌性的雁形目鳥類,較佳係為鴨或鵝,係以一抗原 免疫之’以引發一或數種感興趣之抗體的產生。該抗原包 括但不限定於一微生物(諸如一致病性或非致病性之細 菌、病毒、黴菌、原生動物、線蟲等)、一天然產生或人工 合成之蛋白質(諸如一毒素或荷爾蒙)、一天然產生或人工 合成之募聚、一重組型蛋白質或其等之片段、以及任何 其他有能力刺激抗體產生之物質,與其等之組合。 该抗體可預期由鳥類血清或鳥兒所生之蛋中獲得。然 而,如前所述,由蛋收集該抗體於成本之考量上通常是較 佳的。產㈣禽將IgY及IgY(A Fe)二種異構類型(is〇types) 由血清轉移至該印黃。&學理上’一鴨蛋之印黃中含有約 卜4 mg的igY/ml以及約3-12 mg的 gY(AFc),因此,每 一個蛋可能可提供1 5 - 8 0 m g的I σ γ T w λ 。 Μ 及 45-240 mg 的, 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)。於任何特定之時間内所產 上#〜人丄A 生的印黃體積在數量 上遂超過女全地自該鳥類獲得的血清w 辦夕贫〜 積。此外,卵音抗 肢之卒取可大規模進行而不需大量的投資 ,、 以一選定之抗原免疫一雌禽之技術作、 者所熟知,而不限於使用某一特定之 ”、、热習该項技藝 使用任何型式的免疫作用,其可藉由適卷、式。本發明可 原,其包括皮下、皮内、肌肉、以及:之路徑来接種抗 官内注射。 第11頁 本紙張尺度適用中國國家標準(CNS) M規格(2丨0><297公餐 (請先閱讀背面之注意事項再填寫本頁)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 五、發明説明(10) (請先閱讀背面之注意事項再填寫本頁) 較佳地,一合適之佐劑係與該抗原共同投予,以增強 該免疫反應。詳言之,可單獨使用一劑完全之弗倫氏佐劑, 或任擇地與一劑後續之非完全弗倫氏佐劑組合使用。已發 現一合適之佐劑的使用係可在一長時間内在該免疫化雌禽 的蛋中’冑效率地維持一咼抗體效價,II此使之可能有效 地生產該所欲之抗體。 於免疫作用期間,一雌禽剛開始係於第0天接種該抗 原,而後於一段期間後再度接受該抗原。該初始免疫與第 一次追加(booster)給藥之期間以及個別追加給藥之期間係 取決於該抗原的特殊特性,且較佳係至少為兩週。通常於 初始免疫作用後的1 0週,一大量之對抗該抗原的反應性抗 體係於該雌禽之體内及其所生的蛋中產生。於該雌禽之血 清及蛋中之該對抗該抗原之特殊抗體的存在及效價量可由 此技藝中數種習知方法來確認。 (2)卵黃的部分純化 經濟部智慧財產局員工消費合作社印製 進行部分純化之程序以移除大部分的非水性生物分子 及顆粒’且較佳係移除卵黃中大部分之不相關蛋白質。任 何傳統中可達成此一目的之方法皆可使用於本發明中,該 範例包括PEG、硫酸葡聚糖、或一天然黏膠質(諸如藻膠酸 納、鹿角膠及黃原膠)之使用,以共沈澱該不欲之物質,以 及一水性緩衝液或水之使用以獲得一富含抗體之水性狀態 物質。 於本發明之較佳具體實施例中,首先將該卵黃與蛋白 第12頁 本纸張又度適用中國國家標準(CNS ) A4規格(210X29*7公釐) 587941 A7 B7 五、發明説明(11 ) 分離,而後以蒸餾水清洗之以盡可能移除蛋清。 刺破包覆 卵黃之卵黃磷蛋白膜(vitelline membrane),而你 设將該分離 之卵黃分離部分以大量之水性缓衝液或水稀釋< ,以形成 一卵黃的懸浮物。所收集之卵黃較佳係以約卜0 • 2至約1 :4 〇 v/v之比例的水性緩衝液或蒸餾水稀釋之,且承 文佳係以1 : 5 至約1 : 3 Ο v/v之比例來稀釋之。已有報告指ψ 卬PH值係為 部分純化之步驟期間的一重要因子(Ε· Μ Δ 1 ' • Akita 及 S· Nakai,J· Food Sci·第 57 卷、629 頁( 1 993)) 之最佳回收率而言,pH較佳係設定於5 經濟部智慧財產局員工消費合作社印製 就卵黃抗體 的範圍之間。於 此步驟中,該溫度較理想係位於0°C -60X:範圍 之間。該印 黃的懸浮物係小心地攪拌以形成一均質的混八Λ σ物,而後靜 置一段足以形成水性及非水性層的時間。而% 向後係藉由離心 方式自該水性卵黃懸浮物中移除水不溶性你μ 貝,·其包括非 水性生物分子,諸如脂蛋白、填脂質、固g菜 寺。而後所獲 得之含有抗體的上清液可藉由習知之傾倒、 及取,及其他 方法而與該黏稠之沈澱物分開。 該卵黃上清液可任擇但較佳係進一步以 _ ^ M 一南濃度之非 變性鹽類來處理,以引發抗體的沈澱。可用认 < 廿 ^ J用於卵黃抗體沈 澱之鹽類的例子包括但不限定於 、τ 、Na2S04、 (NH4)2S〇4、KCn、CaCl2、及 MgS〇4。較佳者為 Na2S〇4 及 (NH4)2S〇4。用於沈澱該抗體之鹽類的濃度是重要的,且依 鹽的種類而定’通常係以印黃上清液之最終體積為基礎而 呈一高於15重量%且低於35重量%之量’較佳係位於20〇/〇 及3 0重量%之範圍内。 第13頁 本紙張尺度適用中國國家標準(CNS ) Α4規格(210X 297公釐) (請先閱讀背面之注意事項再填寫本頁)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 Β7 五、發明説明(12 ) (3)免疫親和純化法 如此處所使用的,該’’免疫親和純化法”或,,免疫親和 層析法,,等名詞係為一以該抗體對一特殊之抗原的吸附特 性為基礎之分離類型。此即,於一特定環境下而結合至一 特殊抗原之抗體係於該環境下與未結合之抗體分離。本發 明係欲以該免疫親和純化法之使用來去除不相關之蛋白 質,其包括免疫球蛋白以外的蛋白質及非抗原結合之免疫 球蛋白。更重要的是,本發明之免疫親和純化法之程序意 外地完成了本發明之一個主要目的,即,自包括IgY之全 體卵黃抗體中分離出所欲的IgY(A Fc)抗體。 依據本發明’該免疫親和純化法係藉著應用一由被固 定至一不可溶擔體上之抗原所組成之“抗原基質(antigen matrix)”來進行。該擔體之種類對本發明之免疫親和純化 法而言是不重要的。任何傳統上適用於一抗原共價結合且 對該所欲之抗體及固定於其上之抗原間之交互作用不起反 應的擔體物質均可被使用。該擔體一般係由交聯之瓊脂膠 糖或乂 ~之葡承糖所製成’诸如該可自p h a r爪a c i a講得之 CNBi*-活化之瓊脂糖凝膠(Sepllar〇se) 4B。 於步驟(2)中部分純化的抗體係溶於一 “結合緩衝 液中’且被施加至該抗原基質上,以使之形成固定抗原 及卵η抗體的免疫複合物。任何對該抗原_抗體之交互作用 不起反應且可有效維持該所欲之結合條件的緩衝液系統皆 可用於本發明。該結合緩衝液較佳係選自由磷酸鹽緩衝 第14頁 本紙張尺度適财_家標準(CNS ) Α4ίϋ7Ικ)Χ29^7 (請先閲讀背面之注意事項再填寫本頁) -訂· 經濟部智慧財產局員工消費合作社印製 587941 A7 B7 五、發明説明(T3 ) 液、MES (2-[1^-嗎^林]乙醇石黃酸)緩衝液、以及b丨s_τris緩衝 液所構成之族中,其中在2〇⑺^濃度下之MES緩衝液是最 佳的。 此處所使用之“流經物質,,此名詞係表示流通過該抗原 基質之抗體溶液,其含有大多數的非結合物質。 基於對免疫親和純化法技術之一般性認知,於一離子 強度高於150 mM下’在約7_9之邱值間,形成一免疫複合 物一般是經常被建議的。然而於此條件下,卵黃抗體(特別 是該由一鴨卵黃而來之卵黃抗體)似乎是較不易以最佳之 狀況與被固定於層析擔體上之抗原相結合。具活性之抗體 仍存在於該流經物質中。於本發明之方法中,用於〗g γ (△ Fc)抗體之免疫親和純化法的條件係為獨特的。於本發明 中 由印再所I備之抗體敢適於弱酸及低離子強度的環境 下,即,於pH於4-7之範圍間且低於5〇 之離子強度下結 合。該抗體較佳係於pH值5-6之範圍且更佳係於5.6_5.8之範 圍内,與經固定之抗原相互作用。於本發明所揭示之結合 條件下,於流經物質中,並沒有發現任何可偵測得之活性 抗體。 (4)抗體的沖提 此處所使用之“沖提液”此名詞係A 、 丁、马一可分離被結合 至抗原基質之抗體的化學溶液,其流過兮尸庙甘^ u 抗原基質而包含 一“沖提物”。一般來說,用於破壞卵普浐w > 秋k體-抗原交互作 用之沖提條件係比破壞哺乳動物抗體-抗眉六^ ^ 仇原乂互作用之沖 第15頁 (請先閲讀背面之注意事項再填寫本頁} 、π 經濟部智慧財產局員工消費合作社印製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 五、'發明説明(14 ) 提條件更溫和。 (請先閱讀背面之注意事項再填寫本頁) 一於低於4或高於8之P Η下緩衝化的典型沖提液係適用 以消除IgY(A Fc)-抗原交互作用。然而,通常不建議使用 具有一極端pH值之沖提液,因為此一激烈條件將可能造成 該抗體之抗原結合能力的嚴重喪失。 此外,該含有高濃度之促溶劑的沖提液可被使用於本 發明中。此處所使用之“促溶劑(c h a 〇 t r 〇 p i c a g e n t) ”或“促 溶物(chaotrope),,之名詞係為一可引發一蛋白質分子(諸 如本發明之IgY(AFc)分子)之構形改變的化學物質,因 此,其通常被視為一蛋白質變性劑。一促溶沖提液係藉由 將疏水性結合區域溶於該水性狀態中而干擾該抗原及抗體 間之交互作用。依據本發明,大部分的結合抗體皆可成功 地以任何含有溫和濃度(> 1 Μ)之促溶劑的中性緩衝液來沖 提之。在大多數例子中,在沖提後移除該促溶物將回復天 然的蛋白質結構。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.
使用於免疫親和層析法之沖提液係包括但不限於pH 經濟部智慧財產局員工消費合作社印製 2.3之〇·1 Μ的甘胺酸-HC1、pH 10.0之〇·1 Μ的甘胺酸-HC1、 pH 3.0之6 Μ的呱呢啶-HC1、3.0 Μ的氣化鉀、5 Μ的碘化 鉀、3·5 Μ的氯化鎂、卜3 Μ的硫氰酸胺/硫氰酸鈉以及6 Μ 的尿素。然而對於經回收之抗體的活性而言,一溫和離子 強度且含有促溶物之中性pH緩衝液,諸如緩衝於2〇 mM MES緩衝液(pH 5.8)或20 mM Tris(三羥曱基氨基曱 烷)(ρΗ 7·5)之3 Μ硫氰酸鈉係較佳使用於實施本發明。 所收集之抗體的活化狀態可輕易地藉由諸如一低離子 第16頁 本紙張尺度適用中國國家標準(CNS ) Α4規格(21〇χ297公酱) 經濟部智慧財產局員工消費合作社印製 587941 A7 B7 五、發明説明(16 ) 黃抗體,以獲致一含有該IgY(A Fc)抗體的沖提物。 依據本發明之方法所純化之印黃抗體係為一均質之 IgY(Z\ Fc)。其純度係以非變性之硫酸十二酯鈉之聚丙稀醯 胺凝膠電泳(SDS-PAGE)來檢查,而該所獲得之IgY(AFc) 的分子量係確認為120 kDa。於凝膠中並無發現IgY的污 染物。因為該抗體的多株特性,使得該IgY(A Fc)的等電 點具有一寬廣的範圍(5.2-7.3)。 依據本發明之方法所純化之IgY(A Fc)既不活化該補 體系統且不與哺乳動物血清中的類風濕因子相結合 (rheumatoid factor)。該 IgY(A Fc)與哺乳動物 IgG 間之免 疫交互反應性並不明顯。因此,本發明亦提供一種適用於 臨床及學術研究使用之新類型的抗體。 本發明亦提供依據本發明而製備出之IgY(A Fc)抗體. 的各種臨床及學術研究用途。 例如,本發明中提供一種免疫化一動物(其包括家禽、 家畜類、及寵物)或病人之方法,該方法係藉由對該病人投 予一治療量之本發明的IgY(A Fc)抗體,以保護其等不受 各種不同的病因物質之影響,該病因物質包括微生物,諸 如細菌、病毒、黴菌、原生動物、線蟲等;以及蛋白質的 或非蛋白質的物質,諸如,過敏原、毒素、毒液、荷爾蒙; 或任何其他可引發一免疫反應之免疫原。經純化之IgY(A Fc)抗體較佳係與一藥學上可接受之载劑(諸如,水、生理 食鹽水等)相結合之方式來投予。 本發明之IgY(A Fc)抗體亦可使用於偵測一由人或動 I----11imnr (請先閱讀背面之注意事項再填寫本頁) 訂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 經濟部智慧財產局員工消費合作社印製 A7 五、發明説明(17 ) 物所取得之身體樣本中,諸如體液、組織、細胞萃出物之 感興趣的病因物質,其包括如一致病性或非致病性之生 物,諸如大腸桿菌〜/ζ·)、腸炎沙門氏菌 (心/m⑽e//a以化……勹、以及其他細菌性有機體;一荷爾 蒙,諸如雌性激素、黃體酮、曱狀腺素等;一主要之組織 相合性之複合物抗原等;一腫瘤標記物,諸如α —胎兒蛋 白、前列腺特定抗原等;一疾病狀態標記物,諸如c_反應 性蛋白質、鐵蛋白等。利用依據本發明所獲得之lgY(A Fc) 抗體,一感興趣之病因物質可藉由任何此項技藝中之熟知 的傳統方法,諸如單一放射免疫擴散法(SRID)、免疫電泳 法、放射免疫分析法(RIA)、酶標記免疫吸附分析法 (ELISA)、西方點墨法(WB)、比濁免疫分析法(tia)或顆粒 立曰進式比濁免疫分析法,以定量或定性地偵測之。 基於以上所界定之最佳結合條件,本發明進一步提供 一種用於分析如上述之病因物質之定量或定性的免疫分析 方法,其中一對抗該病因物質之抗體,較佳係為本發明所 純化之IgY(A Fc)抗體,係於最適結合條件下,於該病因 物質存在或不存在之狀況下進行培養。依據本發明,該最 適條件係為於一低於50 mM之離子強度下以及於pH 4_7 犯圍之間。該抗體較佳係被允許於pH 5_6範圍之間,且更 佳係為pH 5.6-5.8範圍之間與該病因物質相互反應。 本發明亦提供一種用於進行上述免疫分析之套組,其 中匕括有包含對一感興趣之病因物質具專一性之抗體的 。式η彳,该抗體較佳係為依本發明所純化之。^八Fc)抗體, 第19頁 本紙張尺細中國^標準(CNS ) M規婦 ----------»-----、訂------· (請先閱讀背面之注意事項再填寫本頁) 587941 Α7 Β7 五、發明説明(18 ) 其中該免疫分析法係於低於50 mM之離子強度且pH在4-7 之條件下進行。該抗體較佳係於pH值在5-6範圍之間, 且更佳係在5 · 6 - 5 · 8之範圍間’與該病因物質交互反應。 當使用於如一單一放射免疫擴散法(SRID)中時,本發 明之IgY(A Fc)抗體可藉由任何傳統方法而被配製於一由 如洋菜、瓊脂膠糖、殿粉、聚丙烯醯胺凝膠等所組成之擔 體培養基中。例如’一擔體培養基可於加熱下,懸浮於一 緩衝溶液中,該IgY(A Fc)抗體係被加入於其中,且該所 形成之物質係被混合在一起。該所形成之溶液係被倒入至 一玻璃平板或倒入至一塑膠容器中,而後冷卻以固化之。 為了供給該欲被檢測之樣品,係於該所形成之凝勝平板上 形成一樣品井。雖然此技術之原理係為熟習該項技藐者所 熟知的,但該IgY(A Fc)抗體-抗原交互作用之物理要件係 比一般之抗體-抗原之交互作用的物理要件更為嚴苛。特別 是該鴨子的IgY(A Fc)抗體係僅於本發明之特定的結合條 件下,結合至抗原且表現沈殿的性質。該特定的結合條件 亦可於其他免疫分析方法(如歐特勒尼(〇Uchterl〇Ily)方法 (MO)及比濁免疫分析法(TIA))中進行,其中一抗原因該 IgY(A Fc)抗體而產生之沈殿或凝集作用係被視為一必須 的步驟。 因此,本發明之一個最佳具體實施例係提供一種用於 抗原之單一放射免疫擴散分析之方法,其包含: (a)製備含有配製於l_5〇mM之鹽濃度且?11約 緩衝溶液中之卵黃抗體的洋菜凝膠; 第20頁 本紙張尺度適财酬家標準(CNS ) A4規格(2似297公釐—)' -- (請先閲讀背面之注意事項再填寫本頁) ••卿· 訂 經濟部智慧財產局員工消費合作社印製 587941 A7 B7 五、發明説明(19 ) (b) 於5玄洋采中挖出數個井’且自井中移父 '、十米·塊; (c) 製備該抗原於磷酸鹽緩衝液生理食骑 義艰中之稀釋 液;以及 ——!.? (請先閲讀背面之注意事項再填寫本頁) (d) 將各抗原稀釋液及樣品吸取至各別的井中; (e) 培養至少24個小時;以及 (f) 量測該免疫沈澱素圈的直徑,其係以該抗原濃度 之對數呈線性相關。 於一商標記免疫吸附分析法(ELISA)或顆粒增進式比 濁免疫分析法中,一置於樣品中之感興趣的抗原係於一足 以與樣品交互作用之量下,被該固定於樹脂測試平板上之 IgY(A Fc)抗體所捕捉,此結果係為該感興趣之抗原存在的 證據,該樹脂測試平板係諸如為聚氯乙烯、聚笨乙烯等, 或一由如聚苯乙烯乳膠、聚酯乳膠、聚氯乙烯、膨土、玻 璃珠等所製成之微細顆粒。於顆粒增進式比濁免疫分析法 中’該被捕捉之抗原而後係直接依據濁度之改變來偵測.。 該濁度之改變係於320-900 nm下量測。於歯標記免疫吸附 分析法(E LIS A)中,該被捕捉之抗原係進一步藉由一綴合有 訊號物質之抗體來偵測。 經濟部智慧財產局員工消費合作社印製 用於實施本發明之較佳具體實施例 下述之實施例係僅為了舉例說明之目的而提出,.並非 用於限制本發明之範疇。 第21頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公酱 587941 心各 A7 五、發明説明(20 ) 貫施例1 :用於刺激特定抗體產生的免疫程序 1 2隻1 6週大之用於生產抗體及蛋之人類 臂"的鴨 (Anas platyrhynchos va「domestica)係分開祓秦表上·、、、 係接受一 1-5 mg/ml之C_反應性蛋白質(CRp; '子 水中純化而得)的初步皮下注射,該配製於pH 7 $ 4 鹽緩衝液中的C-反應性蛋白質係與一等體積之$八外黾 氏佐劑而乳化。該所使用之抗原的濃度一般係於王弗倫 mg/ml的範圍之間。於該初步注射之後,年輕之此 5 兩個星期接受四次之i-5 mg之抗原的外加注^。=禽係每 開始收集蛋,並標記且儲存於4t下,直到進行打=後, 取及純化。於貫驗期間,係每四個星期重複該追加匕的萃 程序。在各追加注射後的第7天進行血液 Π J休m。雜 血液樣品且收集該所得之血清。 -實施例2 :沈澱鴨抗體的物理要件 於此實施例中,係調查pH及鹽類對鴨抗體沈妒 響。瓊脂膠糖粉末係於加熱下,懸浮 L Ά的影 ^ u z Μ之久絲 及氣化鈉濃度變化的磷酸鹽緩衝溶液中。 1 ΡΗ iy 、 5亥5复脂膠糠、、交、、右 係被冷至56°C,此時加入〇.5 ml之含有 ' 儿。 为〇·4 mg/ml之已純 化CRP的抗原溶液,並攪拌之。該溶液 、、、 σ 、 1;r、破傾倒於一塑膠 之容器中且冷卻之。於該平板上之固 " 1同間内製作出鴨子 抗血清用的井。於實施例丨中所收集之 制m斗丁,上 * 仇血清係以使用於 衣備戎平板之W酸鹽緩衝液稀釋丨和 上 ,且將2 // 1之原 抗血清及稀釋的抗血清置於瓊脂膠糖平 双 < 各別的井中。 第22頁 iφφ~' {請先閱讀背面之注意事¾再填寫本頁〕 訂 經濟部智慧財產局員工消費合作社印製 祕用中關家標準( — 587941 A7 B7 五、發明説明(21 ) 在第一組的實驗中,該含有CRP之瓊脂糖凝膠係各別 於pH 4、5、6、7、8及9下被製備。2 // 1之原抗血清及 稀釋的抗血清係置於此等瓊脂膠糖的井中。培養4 8個小時 之後,確定該免疫沈澱素圈之直徑及形狀,如第1表中所 列者。 第1表 不同pH條件下之免疫沈澱素圈之直徑 抗血清之稀釋 於瓊脂膠糖凝膠中之緩衝液 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 如第1表所示,於pH 4.0之瓊脂膠糖平板中沒有發現 免疫沈澱素圈。雖然該免疫沈澱素圈可於pH大於5 · 0之瓊 脂膠糖平板中發現,但當pH值增加到7時,該沈澱素在 形狀上會漸漸地變小。以免疫沈澱素圈之形狀及尺寸為基 礎來看,在pH 5.0可獲得最佳的沈澱效果。該結.果顯示, 鴨抗體在p Η 5.0時適合與一抗原結合。 第23頁 本紙張尺度適用中國國家標準(CNS ) Α4規格(210X297公釐) (請先閱讀背面之注意事項再填寫本頁) n i^i— m、一m ϋ· —ϋ ·ϋ ml _ 經濟部智慧財產局員工消費合作社印製 587941 A7 B7 五、發明説明(22 ) 於第二組的實驗中,除了在pH 5·0下,於20 mM之各 含有0.5、1.0、或1.5 Μ之氯化鈉的磷酸鹽緩衝液中製備 該瓊脂糖凝膠外,重複上述的程序。培養後,該免疫沈澱 素圈比在不含氯化鈉之瓊脂膠糖平板上所顯示的免疫沈澱 素圈更不清楚(資料沒有顯示)。此結果表示在鹽類的存在 下,鴨抗體的沈澱會明顯地受抑制。 同時可一起說明的是,該於pH 5.0下製備於20 mM之 磷酸鹽緩衝液中的瓊脂膠糖平板較適合用於鴨抗體沈澱的 形成。 實施例3 :抗體之效價強度的測定 該實施例1中獲得之抗血清的效價強度係以SRID來 決定之。該含有CRP之瓊脂膠糖平板係使用20. mM之磷 酸鹽緩衝液(pH 5.0)來製備,如實施例2中所述。2 // 1之 於實施例1中所獲得之抗血清係各自置於在平板上挖洞之 分開的井中。培養之後,一免疫沈澱素圈係圍繞該井而形 成,且確定該與置入之抗血清的效價強度有關的圈直徑。 實施例4 :源自鴨子卵黃之抗體的萃取 該由實施例1中之高度免疫性之鴨子所下的蛋中而收 集的卵黃係充分地以一弱的蒸德水水柱清洗之,以藉此移 除蛋清。量測該卵黃的體積,而後與1 〇倍量之所量得的卵 黃體積的蒸餾水充分地混合。該混合物而後係於.4 °c下保 存至少兩個小時,隨後以Hitachi CR22F之離心機於10,000 第24頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) (請先閱讀背面之注意事項再填寫本頁) mi m I ml、一C"Jn n^i ϋ— «in n 經濟部智慧財產局員工消費合作社印製 587941 A7 B7 五、發明説明(23 ) (請先閱讀背面之注意事項再填寫本頁) rpm下離心一個小時。於離心管中係形成一淺淡之上清液 層以及一半固狀之易受擾動層。小心收集該上清液層,將 成粉末狀之硫酸氨加入其中並慢慢攪拌之,直到、終濃度為 每1 00ml之卵黃萃出物中有25 g的硫酸氨,以使得該卵黃 抗體可被完全地鹽析出來。該沈殿物係藉由於4。〇下,以 1 0,00 0 rpm離心30分鐘而收集之。倒出上清液後,該所獲 得之丸粒係再溶解於合適之缓衝液中,且以該緩彳軒液透析 之,以移除該多餘的硫酸氨。聚集該由此程序所獲得之二 十批的粗萃抗體並保存於4°C ’以用於進一步的實驗中。 該所聚集之抗體的效價強度於如實施例3中所述之SRID 分析法測定下,其免疫沈澱素圈的直徑係為6.1 mm。 經濟部智慧財產局員工消費合作社印製 實施例5 :抗原對溴化氰活化之瓊脂膠糖基質的共價附著 一 CRP溶液係製備於0·1 Μ之碳酸鹽緩衝液中,pH為 8.5,且濃度為5 mg/ml。購自Pharmacia之溴化氰活化之 填脂糖凝膠(Sepharose)4B剛開始係以為該基質體積之1 〇 倍量之1 mM冰的HC1清洗之,而後使之於4°C下與基質 體積之2倍量的CRP溶液反應過夜。耦合效率係以耦合蛋 白質的量(於A2 80之單元)除以該初始蛋白質的量(A2 80) 而計算之。結果顯示該C N B r -活化之瓊脂糖凝膠(S e p h a r 〇 s e) 4B的耦合效率係落於80-85%的範圍内。為了稍後的使用, 該抗原基質係在4°C下,以1 : 1 (v/v)之比例懸浮於配製在 20 mM Tris-HCl (pH 8.5)中之 0·5 Μ 的乙醇胺 (ethanolamine)2個小時,以堵絕仍存在之蛋白質作用位 第25頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) 587941 Μ Β7 五、發明説明(24 ) 置。而後,將抗原基質以含有 0.02%之疊氮化鈉的 PBS 清洗之,並儲存於4 °C中。 實施例6 :卵黃抗體之免疫親和純化法 於接下來的實施例6- 8中,該由實施例4所獲得之鴨 抗體以及實施例5中所製備之抗原基質係使用以說明該用 於卵黃抗體之免疫親和性純化法之合適的結合條件。1 ml 之抗原基質係被填裝於一常用的管柱中,且浸泡於第2表 中所列之一結合緩衝液中。該抗原基質係被允許與0.25 ml 之配製於相同之結合緩衝液中的抗體起反應。該抗原基質 係以該結合緩衝液清洗之,直到該流出物係實質上不含蛋 白質為止。所結合的抗體隨後係以4M之呱呢啶-HC1沖提 之,且在完全透析之後,於280 nm下測量其等.之光學密 度。於不同的結合緩衝液中之鴨抗體的結合能力,係以最 終沖提物中之抗體的量來表示之,如接下來之第2表中所 示0 (請先閱讀背面之注意事項再填寫本頁) ——訂------ 經濟部智慧財產局員工消費合作社印製 表 2 第 量 的 體 抗 之 , 上 下 質 件基 條原 同抗 不該 於於 合 結 量 的 體 抗 之 中 物 提 沖 亥 -5 頁 6 2 第 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) 587941 A7 B7五、發明説明(25 ) 丨^ 結合緩衝液 (A 2 8 〇單元X m 1) 20 mM PB,pH 4 0 • 26 20 mM PB,pH 5 0, ,43 20 mM PB,pH 6 0, .39 20 mM PB,pH 7 0· ,32 20 mM PB,pH 8 0_ ,29 20 mM PB, pH 9 0. ,25 可發現一相當大量之鴨抗體係於一弱酸環境下被結 合。 實施例 7 除了將該各測試組之結合緩衝液的pH值調整至5.2、 5 · 4、5.6、或5.8外,重複實施例6之程序。該結果係示於 下列之第3表中。 (請先閱讀背面之注意事項再填寫本頁)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)
訂 %·. 經濟部智慧財產局員工消費合作社印製 表 3 第 量 的 體 抗 , 之 下上 件 質 條基 同原 不抗 於於 合 結 量 的 體 抗 之 中 物 提 沖 該 頁 27 第 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) 587941 A7 B7 五、發明説明(26 ) 結合緩衝液 (A28q單元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 於 第3表中, pH 5 .6-5 .8的環境係顯示最適合用於該 鴨 抗體 的結合。 實 施例 8 除 了將該磷酸 鹽結合緩 衝液置換為一 MES緩衝液或一 Bis-Tri s緩衝液外, 係於 pH 5.8下重複該實施例7的程序。 該 結果 係示於下列 之第 4表 中 。 弟4表 於不 同條件下, 結合於抗原 基質上之抗體的量 該沖提物中之抗體的量 結合緩衝液 (A28〇 單元 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 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 該結果係說明了該調整至pH 5.8之20 mM的MES緩 衝液係為該鴨抗體之免疫親和純化法之最適合的結合緩衝 液。 第28頁 本紙張尺度適用中國國家標準(CNS ) Α4規格(210Χ 297公釐) 587941 A7 B7 五、發明説明(27 ) 實施例9 :沖提效率 除了於沖提液中使用1-3 Μ的硫氰酸鈉(pH 7.5)作為 一促溶劑以與4 Μ之呱呢啶- HC1比較外,藉使用20 mM之 MES緩衝液為該結合緩衝液(pH 5.8)而重複實施例6之程 序。該4 Μ之呱呢啶-HC1的沖提效率在功能上被界定為 1 00%,以作為一參考標準。其他沖提液之相對的沖提效率 係藉由以各沖提液沖提之Α28〇的單元除以4 Μ之呱呢淀 -HC1沖提之Α28〇的單元而決定之。該結果係示於下列之第 5表中。 (請先閲讀背面之注意事項再填寫本頁) 第5表 沖提液的效率 沖提液 效土 4 Μ 呱呢啶-HC1, pH 8.0 100 3 Μ 硫氰酸鈉,p H 7.5 95 2 Μ 硫氰酸納,p H 7.5 70 1 Μ 硫氰酸納,ρ H 7.5 55 ----訂 經濟部智慧財產局員工消費合作社印製 於第5表中可看出,該3 Μ之硫氰酸鈉,其約自該抗 原基質中沖提出約9 5 %的結合抗體,係幾乎與4 Μ之呱呢 啶-HC1的效率相同。 實施例1 0 :以免疫親和純化之於抗原基質上之鴨抗體的純 度及活性 第29頁 本紙張尺度適用中國國家標準(CNS ) Α4規格(210Χ 297公釐) 587941 經 濟 部 智 慧 財 產 局 員 工 消 費 合 社 印 製 A7 B7 五、發明説明(28 )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)
為了檢測該依本發明所製造之抗體的組成,係於8 % 之非還原性之聚丙烯醯胺凝膠上進行分析性的 SDS-PAGE,其中加入60 # g的粗萃抗體以及4〇 ^ §之於 實施例3中所收集的流經物質(各別為第2及第3跑道), 以及加入20 " g之於實施例9中藉由3 M之硫氰酸鈉(pH 5· 8)所沖提之抗體產物(第4跑道)。該結果係顯示於第2 圖中。 該粗萃產物以及該流經物質二者皆顯示兩個明顯的 180 kDa及120 kDa的帶狀(bands)(該等大小係以第工跑 道中之分子重量標記物來估量之),其係各別符合該鴨子之 IgY及IgY(Z\ Fc)的相對移動性。另一方面,親和純化之抗 體係由IgY(A Fc)抗體所構成,其於該凝膠上係顯示一單 一帶狀(band)。此等結果證明,本發明之方法係為一優秀 的策略,而可自該由IgY及IgY(AFc)二者所組成之免疫 球蛋白混合物的聚集中,分離出該所欲的IgY(A Fc)抗體。 幾乎所有的IgY抗體皆不會結合至該抗原基質上,且存在 於該流經物質中。該凝膠之密度分析表示,有超過95%的 純化抗體為均質之IgY(AFC)。此外,存在於該粗萃抗體 之總蛋白質中,有不超過60 %為IgY(AFC)(資料沒有顯 示),且於該粗萃抗體之總蛋白質中的24%(其據信係相當 於該所欲之IgY(AFc)的量)可在親和純化步驟中,結合至 抗原基質上。 該所純化之抗體的活性係藉由於實施例3中所描述之 SRH)來決定。該所純化之抗體的免疫沈殿素圈的直徑係為 第30頁 本紙張尺度適用中國國家標準(CNS ) A4規格( (請先閲讀背面之注意事項再填寫本頁)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 五、發明説明(29 ) ,僅略小於粗萃抗體的6.1 鴨子抗-CRP抗 體 (請先閲讀背面之注意事項再填寫本頁) 實施例11 :以SRID方法決定CRP之濃度 除了以實施例1 0中之親和性純化之 來取代CRP而加入至該違脂膠糖平板中至終濃度到:二 夕卜,以實施例2中所述之使用2〇㈣碟㈣㈣ 液(PH 5.0)來製借王复脂膠糖平板。劑量之來自cRp· 陽性之提供者的人類血清係各自加入至在平板上固定區間 上挖洞之分開的井中,且培養48個小時。測量該免疫沈殿 素圈的直^L 對该CRP標準品之濃度的對數作圖,以獲 得-標準曲線。於整個全部之測量範圍 :係,…圖所示。於未知樣品中的CRp濃度;藉二 才示準曲線之内插法而估算之。該由本發明中之SRID方法 所得之結果係完全與由一購自日本東京之Denka Seiken587941 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 之商業 化CRP-乳膠套組所測得之結果相符(第4圖 實施例12 :以ELISA方法決定CRP之濃度 經濟部智慧財產局員工消費合作社印製 該於實施例1 〇中以親和純化而得之鴨子抗CRP抗體 係以一塗覆緩衝液(c 〇 a t丨n g b u f f e Γ) (2 〇 m μ的填酸鹽緩衝液, ρΗ 8·2)稀釋至終濃度為16/i g/ml。而後,將100 // 1之所 獲得的溶液分配至一 ELISA平板(購自可事達(Costar)股份 有限公司)的各井中。該平板係於4艽下培養過夜或於3 7 °C培養下3個小時。於培養末期,係於各井中加入200 // 1 第31頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) 587941 A7 ______ B7 五、發明説明(30 ) 之阻斷劑(1 %之配製於塗覆緩衝液之脫脂奶粉),以停止 IgY(A Fc)塗覆於平板上。連續稀釋的crP係於室溫下, 與4配製於一反應性緩衝液(MOmM之配製於20mM Tris 緩衝液中的NaCl)中之塗覆鴨抗體交互作用3〇分鐘。而 後’以一清洗緩衝液(含有〇〇5%吐溫(tween)_2〇之反應性 緩衝液)徹底地清洗該平板。一劑稀釋於反應性緩衝液之山 羊-抗-CRP抗體(購自G〇〇d Bi〇tech c〇rp)係加人至各井 中’且該平板係培養3 〇分鐘,而後以該清洗緩衝液徹底清 洗之。接下來,一劑稀釋於反應性緩衝液之HRP綴合之兔 子-抗-山羊IgG(Sigma)係被加入至各井中,且該平板係於 室溫下培養30分鐘,而後以該清洗緩衝液徹底清洗之。1 〇〇 // 1之〇 -本一胺二氣化氫(S i g ni a)係被加入至各井中’以 作為該綴合酵素的受質,該平板係於室溫下培養1 5分鐘。 該反應係以1 00 // 1之1N的H2S04來中和。測量各井於 4 5 Onm下的吸收值,並繪製於第5圖中。該測試的敏感度 可高達 2.5 ng/ml (O.D. > 1)。R-平方可高達 0.9941。 實施例1 3 :以顆粒增進式的τιA方法(LAT :乳膠免疫比 濁分析法)決定CRP之濃度 將抗-CRP IgY( △ Fc)耦合至該乳膠粒子上之梦驟係在 4°C下,於一含有1〇〇 # 1之ίο% (w/v)乳膠懸浮物、400 // 1之20 mM Tris緩衝液(ρΗ7·2)、10 mg/ml之由實批例 10所純化之鴨子抗-CRP抗體、以及0. 1 %之疊氮化納的反 t θ I白 應混合物中進行2個小時。5 ml之含有1 %牛血μ @ 丨!二 :G lu :| Γ : ----------------11------ (請先閲讀背面之注意事項再填寫本頁) :_ 經濟部智慧財產局員工消費合作社印製 第32頁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
本紙張尺度適用中國國家標準(CNS ) Α4規格(210X297公釐) 587941 A7 B7 經濟部智慧財產局員工消費合作社印製 五、發明説明(31 ) (BSA ;購自Sigma)的20 mM Tris緩衝液(pH7.2)係被加入 至該乳膠-IgY( △ Fc)混合物中,且該所獲得之懸浮液係於4 °C培養1.5個小時,並攪拌之。於4°C下以1 5,0〇〇 rpm離 心1個小時後,該所收集之丸粒係再懸浮於5 ml之含有 1%BSA的20 mM Tr1S緩衝液(PH7.2)中.,以供後續使用。 該含有CRP之校準物血清係被作為一標準抗原,於高 範圍下之澴度為:〇、1、5、10、及20 nig/di,而於低範圍 下之濃度為:0、0.5、1、1.5、及21^/(11。兩種添加劑係 為R1 : 20 mM Tris緩衝液(pH 7.2)以及R2 :懸浮於R1中 之IgY( △ Fc)-雜合之乳膠懸浮物。該因R2與標準抗原混 合而導至之吸收值(於5 70 nm)的增加係以Hitachi 7020之 比濁自動分析儀對應時間而進行測量,且所獲得之數據係 各別對該低範圍及高範圍之CRP濃度作圖,如第6a及6b 圖所示。因此’於未知樣品中之CRP濃度可藉由該標準曲 線之内插法而估算之。 於本發明之說明書中所引述之所有的專利及文獻參考 資料係完整地併入於此,以作為參考資料。若與本案有矛 盾之處,則以本發明之說明(包括定義)為準。 同時本發明亦已利用上述之特殊實施例予以說明,必 須了解的是’對熟習该項技勢者而言係為顯知之各種不同 的改良及變化可在不偏離本發明之精神及範嘴下進行。 第33頁 本紙張尺度適用中國國家標準(CNS ) A4規格(210X297公釐) LT—! (請先閱讀背面之注意事項再填寫本頁) ΗThis 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
mu If mi-ί-r^Jϋϋ mi —ϋ —.ϋ ml n 一>.>:* * t tl ,mu If mi-ί-r ^ Jϋϋ mi —ϋ —.ϋ ml n 一 >. >: * * t tl,
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