TW562925B

TW562925B - Immunogenic compositions for use in immunoassay methods to measure diacyl hydrazines or derivatives thereof and antibodies raised by the same, and methods and kits of measuring diacyl hydrazines or derivatives thereof

Info

Publication number: TW562925B
Application number: TW86111727A
Authority: TW
Inventors: James Douglas Thacker; Ellen Schalk Casale; Stanley Stephen Stavinski; Shuguang Wu
Original assignee: Dow Agrosciences Llc
Priority date: 1996-08-12
Filing date: 1997-09-17
Publication date: 2003-11-21
Also published as: JPH10310536A; HUP9701386A3; HU9701386D0; HUP9701386A2; ZA977188B

Abstract

The present invention provides an immunogen, antibodies, kits and methods of using the same to measure diacyl hydrazine compounds. The methods are easy to use, inexpensive and provide suitable cross-activity and sensitivity to enable use under FIFRA guidelines.

Description

62925 A7 B7 經濟部中央標準局員工消費合作社印製五、發明説明（1 ) 本發明係&供一種測足一 s蠢基骄化合物的免疫分析方法。測足一醯基胼化合物的方法是已知的。例如，高壓液相層析法（HPLC)、氣液相層析法（GLC)、GC(氣相層析）-質讀分析和HPLC質譜分析是目前僅有少數可利用的方法。然而，這些方法有數個缺點。這些方法通常需要筇貴和複雜的儀器。再者，試樣製備和實際的分析可能是相當冗長的，並且需要接受過這類方法之特別訓練的化學家和技術員。此外’這類先前技藝的方法在相同的步驟中不能迅速地篩選出類似物或代謝物或兩者。更確切地説，在檢測發生之前通常需要分離個別的化合物。結果，這些方法不能應付迅速低價之測定的需求，例如野外測試或大量體積之測試。發現二酿基胼化合物作爲除害劑是特別有用的。更特定而呂，係作爲毛蟲蜕皮加速化合物（M A c )。就像所有可能的危險化學物質，藉著政府實體來規範這類除害劑。在獲知規足批准的過程中，可能需要在聯邦殺蟲劑、殺眞菌劑和滅菌劑作用（Federal lnsecticide，Fungicide and Rodenticide Act) ( FIFRA)註册指導方針〇部份（Registrati〇n Guidelines，Subdivision 〇)之下的殘餘物分析，需要具有適合這類用途的適當敏感性之分析。再者，該分析必須具有與二醯基肼除害劑之主要代謝產物的適當交叉反應性，以便適用於在FIFRA註册指導方針N部份中所需的代謝和環境歷程研究。爲了監視該除害劑的使用也可能需要該分標準(CNS ) A4· (請先閱讀背面之注意事項^^寫本頁)62925 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the Invention (1) The present invention is an immunoassay method for measuring a compound based on amylolamine. A method for measuring a fluorenyl amidine compound is known. For example, high-pressure liquid chromatography (HPLC), gas-liquid chromatography (GLC), GC (gas chromatography) -mass analysis, and HPLC mass spectrometry are currently only a few methods available. However, these methods have several disadvantages. These methods often require expensive and complex equipment. Furthermore, sample preparation and actual analysis can be quite lengthy and require specially trained chemists and technicians with such methods. In addition, such prior art methods cannot quickly screen for analogs or metabolites or both in the same step. Rather, it is often necessary to isolate individual compounds before detection occurs. As a result, these methods cannot cope with the need for rapid, low-cost measurements, such as field tests or large volume tests. Dimeryl amidine compounds have been found to be particularly useful as pesticides. More specifically, it is a caterpillar molting accelerating compound (M A c). Like all possible hazardous chemicals, this type of pesticide is regulated by government entities. In the process of obtaining regulatory approvals, it may be necessary to register in the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) Registration Guidelines Part 0 (RegistratiOn Guidelines, Subdivision Analysis of residues below 0) requires analysis with appropriate sensitivity suitable for this type of use. Furthermore, the analysis must have appropriate cross-reactivity with the major metabolites of the dihydrazide pesticides to be suitable for the metabolic and environmental history studies required in Part N of the FIFRA registration guidelines. This sub-standard (CNS) A4 may also be required in order to monitor the use of the pesticide (please read the precautions on the back first ^ write this page)

訂Order

^62925 A7 B7 五、發明説明（析結果，需要一種具有滿足FIFRA指導·方釙之敏感性和交叉反應性、能夠迅速篩選，並在使用上是簡單和價廉的二醯基胼分析。本發明已經發展一種免疫化學分析；它是充份敏感的並具有足以在FIFRA指導方針下使用的交叉反應性。此外，該分析在使用上是簡單且價廉的。“ 一在本發明的第一個觀點中，係提供一種免疫原，包括：下式之化合物 - 0 fl (請先閲讀背面之注意事丨丨本頁^ 62925 A7 B7 V. Explanation of the invention (Analytical results, a sensitivity and cross-reactivity that meet FIFRA guidelines and side effects, can be quickly screened, and is simple and inexpensive to use. The invention has developed an immunochemical analysis; it is sufficiently sensitive and cross-reactive enough to be used under the FIFRA guidelines. Furthermore, the analysis is simple and inexpensive to use. "One in the first of the invention In one aspect, an immunogen is provided, including: a compound of the formula-0 fl (please read the notes on the back first 丨丨 this page

、1Τ 經濟部中央標準局員工消費合作杜印製其中R、R1、R2、R3和R4分別爲氫、（Ci-C6)烷基和經取代的（CVC6)烷基、（C2-C6)^基和尨取代的（C2-C6)烯基、（CrCJ烷氧基、（CrCd烷氧酸基和齒素；連結到一載劑物質。在本發明的第二個觀點中，係提供一種測定二醯基胼或, 1T Consumer Cooperation of the Central Standards Bureau of the Ministry of Economic Affairs, where R, R1, R2, R3 and R4 are hydrogen, (Ci-C6) alkyl and substituted (CVC6) alkyl, (C2-C6) ^ And fluorene-substituted (C2-C6) alkenyl, (CrCJ alkoxy, (CrCd alkoxy acid, and dentin); linked to a carrier substance. In a second aspect of the present invention, an assay is provided Dioxin

I 其衍生物的方法，包括下列步驟··（ A)提供含有.未知含量 \ 之二醯基胼的試樣；（B )使該試樣、在固定不動之二醯基胼抗原的存在下，與對二醯基肼抗原具有結合親和力的抗體接觸，以致於形成結合和未結合之抗體-抗原複合物；（C ) 從已結合之抗體-抗原複合物中分離未結合的抗體-抗原複 5- ΜΛ張尺度適用中國國家標準（CNS )八4^格（210X297公釐） 562925 A7 _____ B7 五、發明説明（3 ) 合物；（D )以可檢測之標記來標示已結合的抗體複合物； (E)引起該標記中可測量的變化；並（F)測定在該試樣中之二醯基胼。在本發明的第三個觀點中，係提供一種測定二醯基胼或其衍生物的方法，包括下列步驟：（A)提供含有未知含量之二醯基胼的試樣；（B)使該試樣與對二醯基肼抗原具有結合親和力之固定不動的抗體接觸，形成固定不動的抗體_抗原複合物；（C)使利用可檢測之標記標示的二醯基肼抗原與未複合之固定不動的抗體接觸，形成經過標示之抗體_抗原複合物，（D)引起該標記中可測量的變化；並（e )測定在該試樣中之二醯基肼。在本發明的第四個觀點中，係提供一種測定二醯基肼的工具組，包括（A)對二醯基胼具有結合親和力之抗體；（B) 能夠與該抗體結合的標記；以及（c)在該標記的存在下能夠產生可測量之變化的受酶質。圖式簡單說明圖（1)描述得自DEAE離子交換管柱之蛋白質洗脫側面圖。圖（2)描述RH2703之抑制曲線。圖（3)描述RH2651之抑制曲線。圖（4)描述RH5992之抑制曲線。圖（5)描述RH0345之抑制曲線。圖（6)描述RH2485之抑制曲線。圖（7)描述在P B S T和基質空白試樣中獲得的外標準曲 -- " - ' ' --—--— - ft - _. 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公釐) ---—--- 經濟部中央標準局員工消費合作社印製 562925 A7 B7 五、發明説明（圖（8)描述關於硬花甘藍（Br〇cc〇H)試樣2〇139_〇丨的計算線性回歸線。圖（9)描述關於硬花甘藍試樣2〇139_〇2的計算線性回歸線。圖（ίο)描述關於硬花甘藍試樣20139-04的計算線性回歸線0 圖（1 1)描述關於硬花甘藍試樣2〇139_〇5的計算線性回歸線。圖（1 2 )描述關於土壤試樣15 i的計算線性回歸線。圖（1 3 )描述關於土壤試樣157的計算線性回歸線。圖（1 4 )描述關於土壤試樣丨75的計算線性回歸線。圖（1 5 )描述關於土壤試樣丨87的計算線性回歸線。圖（1 6) a和b描述直接分析曲線和直接分析標準曲線。當在本文中使用”抗原"一詞時，明瞭其爲任何能夠刺激柷體產生的物質。同樣地，明瞭”二醯基肼抗原”爲任何能夠刺激抗體產生的二醯基胼化合物，其中所產生的抗體具有對一酿基胼及其衍生物的結合親和力。此外，亦明瞭，，免疫原一’包括任何用來誘發免疫反應的物質。免疫原通常包括載劑分子和其他成份（半抗原）。當在文中使用”測定”一詞時，明瞭其包含定性分析，也就是確認分析物存在於試樣中，以及定量分析，也就是定出該分析物在試樣中的含量。亦明白這類名詞可包含標準物和標準曲線之製備，這類製備作用是此項技藝中已熟知的。本紙張尺度適用中國國家標準（CNS )八4規格（21〇χ297公釐I A method of its derivatives, including the following steps: (A) providing a sample containing an unknown amount of amidinofluorene; (B) allowing the sample to be immobilized in the presence of a fixed amidinofluorene antigen Contact with an antibody that has binding affinity for the dihydrazide antigen, so that a bound and unbound antibody-antigen complex is formed; (C) separating the unbound antibody-antigen complex from the bound antibody-antigen complex 5- ΜΛ Zhang scale is applicable to the Chinese National Standard (CNS) 8 4 ^ grid (210X297 mm) 562925 A7 _____ B7 V. Description of the invention (3) Compound; (D) Detected label to indicate the bound antibody complex (E) cause a measurable change in the label; and (F) determine the two fluorene radicals in the sample. In a third aspect of the present invention, a method for determining difluorenylfluorene or a derivative thereof is provided, including the following steps: (A) providing a sample containing an unknown content of difluorenylfluorene; (B) making the The sample contacts the immobilized antibody with binding affinity for the dihydrazide antigen to form an immobilized antibody-antigen complex; (C) the dihydrazide antigen labeled with a detectable label and the uncomplexed immobilized Immobilized antibodies are contacted to form a labeled antibody-antigen complex, (D) causing a measurable change in the label; and (e) determining the dihydrazine in the sample. In a fourth aspect of the present invention, there is provided a tool set for determining dihydrazine, comprising (A) an antibody having binding affinity for difluorenylhydrazone; (B) a label capable of binding to the antibody; c) Enzyme capable of producing measurable changes in the presence of the label. Brief description of the figure Figure (1) depicts a side view of the protein elution from a DEAE ion exchange column. Figure (2) describes the inhibition curve of RH2703. Figure (3) describes the inhibition curve of RH2651. Figure (4) describes the inhibition curve of RH5992. Figure (5) depicts the inhibition curve of RH0345. Figure (6) describes the inhibition curve of RH2485. Figure (7) describes the external standard curve obtained in the PBST and matrix blank samples-"-'' -------ft-_. This paper size applies to China National Standard (CNS) A4 specifications (210X297 (Mm) -------- Printed by the Employees' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 562925 A7 B7 V. Description of the invention (Figure (8) describes the broccoli (Brocco) sample 2〇139_ 〇丨 Calculated linear regression line. Figure (9) describes the calculated linear regression line on the broccoli sample 2〇139_〇2. Figure (ίο) describes the calculated linear regression line on the broccoli sample 20139-04. 1 1) Describe the calculated linear regression line for broccoli sample 2013_05. Figure (1 2) describes the calculated linear regression line for soil sample 15 i. Figure (1 3) describes the soil sample 157. Calculate the linear regression line. Figure (1 4) describes the calculated linear regression line on the soil sample 75. Figure (1 5) describes the calculated linear regression line on the soil sample 87. Figure (16) a and b describe the direct analysis curve. And direct analysis of the standard curve. When using the term "antigen" in this article, it is clear It is any substance that can stimulate carcass production. Similarly, it is clear that "dihydrazide antigen" is any dihydrazone compound that can stimulate the production of antibodies. Binding affinity. In addition, it is also clear that an immunogen includes any substance used to induce an immune response. Immunogens usually include carrier molecules and other components (haptens). When the term "assay" is used in the text, it is clear It includes qualitative analysis, that is, confirming the presence of the analyte in the sample, and quantitative analysis, that is, determining the content of the analyte in the sample. It is also understood that such terms can include the preparation of standards and calibration curves. This kind of preparation is well known in the art. This paper size is applicable to China National Standard (CNS) 8 4 (21 × 297 mm)

562925 A7 B7 五、發明説明（5 經濟部中央標準局員工消費合作社印製 ‘在本文中使用’’二酸基耕化合物” 一詞時，明瞭在其範圍内包括二醯基胼以及從其中衍生的代謝產物、合成的類似物、環境降解產物和光化學降解產物。當在本文中使用” I c 5〇” 一詞時，係限定爲減少吸光度至在/又有抑制劑存在時測得之吸光度的一半，所需之抑制劑的濃度。該測定係藉著使殘留活性的百分比（％)對抑制劑濃度作圖來完成，藉著活性％ =(八。-八；/八〇) X 100 知到殘留的活性％，其中A。爲在沒有抑制劑存在時測得的吸光度’而八丨爲在ith之抑制劑濃度時測得的吸光度。然後藉著100-(活性％)的關係式得到抑制百分比。藉著等式導出交又反應性百分比：叉又-反應性。/〇=(IC5。5992/IC5() ana丨。g)xl00 其中ICw，5992爲按照上文測定之RH5992的IC5〇濃度，而 ic5〇，ana丨。§爲類似物，也就srH27〇3、rH2651 *rH〇345 的 I C 5 0濃度。按照在等式中的描述·· S = (Yint/m)xV 從得自標準濃度之重覆分析的標準偏差之回歸分析測定的抑制劑濃度來計算以毫微克計之敏感性，其中S爲以毫微克計l敏感性，Yint爲以吸光度單位計，得自重複分析之標準偏差的回歸線的γ·截取値，m爲以每毫微克/毫升之吸光度單位計的回歸線斜率，且V爲以毫升計，應用在微量滴定上的試樣體’積。從關係式：％& = ((：。/(^)父1〇〇 (請先閲讀背面之注意事本頁) ......---1 1- · 4 --. 太訂 8- 本紙張A度適用中國國家標隼（CNS ) A4規格（210X297公釐 562925 6 經濟部中央標準局員工消費合作社印製 A7 B7 五、發明説明（來計算回收百分比，其中C。爲觀察到的（測得的）濃度，而 C a爲實際的（加料的）濃度。如同上文詳述的，提供可用來製備對二醯基肼之抗體的免疫原。通常該免疫原包括半抗原和载基分子。半抗原通常是二醯基肼分子或其衍生物。在一個具體實施例中，該半抗原是苯甲醯胼。苯甲醯肼化合物是根據上文是（1)之化合物。 (C ! - C6)烷基的適當實例包括但不限於甲基、乙基、正· 丙基、異丙基、正-丁基、異丁基、第三-丁基、正-戊基、異戊基、新戊基、正-己基、異己基等等。（C^Cs)經取代燒基之適當實例包括但不限於以基、自素、（C i- c 4) :¾ 氧基和硝基取代的甲基、乙基、正-丙基、異丙基、正_丁基、異丁基、第三-丁基、正-戊基、異戊基、新戊基、正_ 己基等等。 (C C6)烯基的適當實例包括但不限於乙烯基、正-丙缔基、異丙烯基、正· 丁烯基、異丁烯基、第三_ 丁晞基、正· 戊烯基、異戊烯基、新戊烯基、正-己烯基等等。（C c ) 經取代之烯基的適當實例包括但不限於以羥基、自素或硝基取代的乙缔基、正-丙歸基、異丙烯基、正_ 丁缔基、異丁烯基、第三_ 丁烯基、正-戊晞基、異戊晞基、新戊烯基、正-己晞基等等。 (c i- C6)烷氧基之適當實例包括但不限於曱氧基、乙氧基、正-丙氧基、異丙氧基、正-丁氧基、異丁氧基、第= 丁乳基、正-戊氧基、異戊乳基、新戊氧基和正-己氧基。 9 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）562925 A7 B7 V. Description of the invention (5 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs' when using the term 'di-acid-based cultivation compound' in this article, it is clear that the scope includes dihydrazone-based hydrazone and derivatives derived therefrom Metabolites, synthetic analogs, environmental degradation products, and photochemical degradation products. When the term "I c 50" is used herein, it is limited to reducing the absorbance to the absorbance measured in the presence of an inhibitor. The required concentration of inhibitor. This determination is done by plotting the percentage of residual activity (%) against the concentration of inhibitor, by% of activity = (eight.-eight; /eight.80) X 100 The residual activity% is known, where A. is the absorbance measured in the absence of an inhibitor 'and 丨 is the absorbance measured at the inhibitor concentration of ith. Then, by the relationship of 100-(% of activity) The percent inhibition is obtained. The percent reactivity is derived by the equation: cross-reactivity. / 〇 = (IC5.5992 / IC5 () ana 丨 .g) xl00 where ICw, 5992 is the RH5992 determined according to the above. IC50, and ic50, a na 丨. § is an analog, that is, the IC 50 concentration of srH27〇3, rH2651 * rH〇345. As described in the equation ... S = (Yint / m) xV from the repetition of the standard concentration The standard deviation of the analysis is determined by regression analysis of the inhibitor concentration to calculate the sensitivity in nanograms, where S is the sensitivity in nanograms and Yint is in absorbance units, derived from the regression line of the standard deviation of the repeated analysis γ · cutting 値, m is the slope of the regression line in units of absorbance per nanogram / ml, and V is the product of the sample body applied in micro titration in ml. From the relationship:% & = (( ： ./ (^) Father 1〇〇 (Please read the note on the back page first) ......--- 1 1- · 4-. Taidang 8- This paper is applicable to Chinese national standard A隼 (CNS) A4 specification (210X297 mm 562925 6 Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of the invention (to calculate the recovery percentage, where C. is the observed (measured) concentration, and C a is the actual (additive) concentration. As detailed above, provides a resistance to dihydrazide The immunogen usually includes a hapten and a carrier molecule. The hapten is usually a dihydrazine molecule or a derivative thereof. In a specific embodiment, the hapten is benzamidine. Benzoazine The compound is a compound according to (1) above. Suitable examples of (C! -C6) alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl , Tertiary-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl and the like. Suitable examples of (C ^ Cs) substituted alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso Propyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like. Suitable examples of (C C6) alkenyl include, but are not limited to, vinyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, tertiary-butenyl, n-pentenyl, isopentenyl , Neopentenyl, n-hexenyl, and the like. (C c) Suitable examples of substituted alkenyl include, but are not limited to, ethylenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl, n-butenyl, Tri-butenyl, n-pentamyl, isopentyl, neopentenyl, n-hexyl and the like. Suitable examples of (c i-C6) alkoxy include, but are not limited to, fluorenyloxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, thyryl Group, n-pentyloxy, isoamyloxy, neopentyloxy and n-hexyloxy. 9 This paper size applies to China National Standard (CNS) A4 (210X297 mm)

562925 、發明説明（ (ci-C6)烷氧酸基的適當實例括但不限於複基卜 (請先閲讀背面之注意事 COOH)、乙酸基（-CH2COOH)、丙酸基（-CH2CH2COOH) 和正·丁酸基（-ch2ch2ch2cooh)。 i素的適當實例包括但不限於cr、ΒΓ-、F·和Γ。在較佳的具體實施例中，：^爲πΗΑΟΟΗ，R1和R2分別爲甲基，且R3和R4分別爲氫。在更佳的具體實施例中，R 馬-COOH，R1和R2分別爲甲基，且RqpR4分列爲氫。載劑物質可以是蛋白質，舉例而非限制：牛血清白蛋白、卵清蛋白或鎖孔貝（keyhole limpet)血藍蛋白；多醣，舉例而非限制：葡聚糖、瓊脂糖、瓊脂糖或纖維素；或是石成的聚合物或共聚物，舉例而非限制：聚丙晞遂、聚醯、1Τ 胺、聚丙烯醯胺、聚丁酸酯、聚脲、聚脲醯胺或聚苯乙烯 0 在較佳的具體實施例中，該载劑物質爲載劑蛋白質，較佳的是牛血清白蛋白（BSA)或鎖孔貝血藍蛋白（KLH)，最佳的是鎖孔貝血藍蛋白。562925, suitable examples of the invention description ((ci-C6) alkoxy acid group) include but are not limited to compound base (please read the note on the back COOH), acetate (-CH2COOH), propionate (-CH2CH2COOH) and n- Butyric group (-ch2ch2ch2cooh). Suitable examples of i-primes include, but are not limited to, cr, β-, F, and Γ. In a preferred embodiment, ^ is πΗΑΟΗ, R1 and R2 are methyl, And R3 and R4 are hydrogen respectively. In a more specific embodiment, R MA-COOH, R1 and R2 are methyl groups, and RqpR4 is classified as hydrogen. The carrier substance may be a protein, for example and not limitation: cattle Serum albumin, ovalbumin, or keyhole limpet hemocyanin; polysaccharides, by way of example and not limitation: dextran, agarose, agarose, or cellulose; or stone-formed polymers or copolymers, for example Without limitation: polypropylene, polyfluorene, 1T amine, polyacrylamide, polybutyrate, polyurea, polyuretamine, or polystyrene. In a preferred embodiment, the carrier substance is Carrier protein, preferably bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), Jia Pui is keyhole limpet hemocyanin.

可以利用本發明之免疫原來製備對二醯基肼化合物的抗體。將該免疫原導入適當的動物體内，並收集、分離和純經濟部中央標準局員工消費合作社印製化抗體。所得的抗體是多株抗體，對二醯基肼化合物及其衍生物具有高親和力和交又反應性，特別是苯甲醯肼及其衍生物。 .另外，可以經由使用此項技藝中已熟知的融合瘤技術，利用該免疫原來製備單株抗體。在一個具體貫施例中，與載劑物質連結的式（i)免疫原使 10- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公董） 562925 五、發明説明（8 抗體昇高。在較佳的具體實施例中，與載劑物質連紝，直中R爲.ch3COOH，r>r2爲甲基，r^r4爲氯的式㈠ ^原，使抗體昇高。在更佳的具體實施例中，與載劑物質連結，其中Rfc〇OH , Rl和R2爲甲基，R3和式（1)免疫原，使抗體昇高。同上文詳述的，本發明提供一種測定二醯基胼或其衍生物的万法。最初，該方法包括(A)提供包含未知含量之二酿基耕的試樣之步驟。試樣通常是任何可能含有二醯基胼的物質。適當的實例包括空氣、水、土壤或生物物質。在一個具體實施例中，試樣可以是空氣試樣或衍生自空氣試樣的試樣。在另一個具體實施例中，試樣可以是水試樣或衍生自水試樣的試樣。在另一個具體實施例中，試樣可以是土壤試樣或衍生自土壤試樣的試樣。經濟部中央標準局員工消費合作社印製在一個具體實施例中，試樣可以是生物物質或含有生物物質的試樣，或是衍生自生物物質之試樣。生物物質之適當實例包括但不限於植物物質；諸如血液、血清、血漿、淋巴液、胃灌洗液、膽汁、玻璃體液之類的生物液；諸如植物和動物組織之類的生物組織，以及諸如細菌和病毒之類的微生物樣本。在較佳的具體實施例中，該試樣爲土壤或植物物質，或從其中衍生的試樣。二醯基胼化合物是如同上文在式1中描述的，及其衍生物。這類衍生物可以是，而非限定於從其中衍生的代謝產 -11- 本紙張尺度適用中國國家標準（CNS ) A4娜（210X297公釐）經濟部中央標準局員工消費合作社印製 562925 A7 B7 五、發明説明（9 ) 物、合成的類似物、環境降解產物、光學降解產物。特別有用的二醯基肼是根據式1之苯甲醯肼，在下列的表1中詳述之。表1The immunogen of the present invention can be used to prepare an antibody against a dihydrazide compound. The immunogen was introduced into appropriate animals, and antibodies were collected, isolated, and purified by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. The obtained antibodies are multiple strains of antibodies and have high affinity and cross-reactivity for dihydrazine compounds and their derivatives, especially benzamidine and its derivatives. In addition, a monoclonal antibody can be produced by using the immunogen by using the fusion tumor technology well known in the art. In a specific embodiment, the immunogen of formula (i) linked to the carrier substance 10- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 public directors) 562925 V. Description of the invention (8 Antibodies Elevated. In a preferred embodiment, it is linked to a carrier substance, where R is .ch3COOH, r > r2 is methyl, and r ^ r4 is chlorine, so that the antibody is raised. In a more specific embodiment, it is linked with a carrier substance, wherein RfcoOH, R1 and R2 are methyl groups, and R3 and the immunogen of formula (1) make the antibody elevated. As detailed above, the present invention provides a Method for the determination of difluorenylpyrene or its derivatives. Initially, the method included (A) a step of providing a sample containing an unknown amount of Nitrogen. The sample is usually any substance that may contain difluorenylpyrene. Suitable examples include air, water, soil, or biological matter. In a specific embodiment, the sample may be an air sample or a sample derived from an air sample. In another embodiment, the sample may be water Or a sample derived from a water sample. In another embodiment, The sample can be a soil sample or a sample derived from a soil sample. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs In a specific embodiment, the sample can be a biological substance or a sample containing a biological substance, or it can be derived Samples from biological matter. Suitable examples of biological matter include, but are not limited to, plant matter; biological fluids such as blood, serum, plasma, lymph fluid, gastric lavage fluid, bile, vitreous body fluids; such as plant and animal tissue Biological tissues, and microbial samples such as bacteria and viruses. In a preferred embodiment, the sample is soil or plant material, or a sample derived therefrom. The dihydrazone compound is as above The text is described in Formula 1, and its derivatives. Such derivatives can be, but are not limited to, the metabolites derived from them. 11- This paper applies the Chinese National Standard (CNS) A4 Na (210X297 mm). Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 562925 A7 B7 V. Description of Invention (9) Objects, synthetic analogues, environmental degradation products, optical degradation products Particularly useful is a two acyl hydrazine of formula 1 of benzoyl hydrazine, described in the following detailed in Table 1. TABLE 1

苯曱醯肼 R R1 R2 R3 R4 RH5992 -CH2CH3 -ch3 -ch3 H H RH2485 Η -ch3 -ch3 -ch3 -och3 RH2703 -CH2COOH -ch3 -ch3 H H RH2651 -COOH -ch3 -ch3 H H RH0345 -Cl H H H H 使步驟（A)的試樣在步驟（B)中，在固定不動的二醯基月井抗原的存在下，與對二醯基肼具有結合親和力的抗體接觸。抗體如同上文的描述，由含有RH2651之免疫原，最好是與KLH連結的，使該抗體昇高。固定不動之二醯基胼抗原中的抗原，可以是任何上述的二醯基胼，與任何上述的載劑分子連結。在較佳的具體實施例中，該固定抗原爲RH2651或RH2703，與載劑蛋白質連結，較佳的是與B S A連結的RH2703。抗原與載劑分子的組合被稱爲塗覆抗原。塗覆抗原一般被固定在具有表面的固體支撑物上，該表面負責附接這類抗原。適當的實例包括但不限於微量滴定 -12- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）Phenylhydrazine R R1 R2 R3 R4 RH5992 -CH2CH3 -ch3 -ch3 HH RH2485 Η -ch3 -ch3 -ch3 -och3 RH2703 -CH2COOH -ch3 -ch3 HH RH2651 -COOH -ch3 -ch3 HH RH0345 -Cl HHHH Make step ( A) The sample in step (B) is contacted with an antibody having binding affinity for dihydrazine in the presence of immobilized dihydrazyl moonwell antigen. As described above, the antibody is raised by an immunogen containing RH2651, preferably KLH. The antigen in the immobilized amidinofluorene antigen may be any of the aforementioned amidinofluorenes, which is linked to any of the aforementioned carrier molecules. In a preferred embodiment, the immobilized antigen is RH2651 or RH2703, which is linked to a carrier protein, preferably RH2703 linked to B S A. The combination of antigen and carrier molecule is called a coated antigen. Coated antigens are typically immobilized on a solid support with a surface that is responsible for attaching such antigens. Appropriate examples include, but are not limited to, microtitration. -12- This paper size applies to China National Standard (CNS) A4 (210X297 mm)

562925 五、發明説明（10 盤；諸如硝基纖維素、纖维夸、 :確的γ办仏素纖、准素乙鉍酯、聚碳酸酯卿ί 珠、管柱填充材料之類的東、粒，以及具有結合功能部，彡並卜的圹4主肊夠、…泛抗原使其附接於八上的竹生表面。通當無漁皇覆抗原塗抹在固體支撑物上並吸附於其上。人碎初上卫在一個具體實施例中，使塗覆抗原吸附在微量滴定盤上0 = :(B)中’在自由抗原，也就是在試樣中未知含量的冬甲酬，和固定抗原，也就是已結合抗原之間，發生了 y有-SS基肼（$親和力的抗體上對於結合位置的競 f -果形成已結合和未結合的抗原_抗體複合物。在步驟中’從未結合的複合物中分離出已結合的複合物。可 I曰著此項技藝中已熟知的任何方法來分離之，像是但不限於=分離、磁性分離，並沖洗以移除未結合的複合物。· 一旦從未結合的抗原_抗體複合物中分離出，便在步驟 (D)^利用可檢測的標記來標示已結合的複合物。該標記可以是任何能夠固定在已結合之抗原-抗體複合物上，並可被檢測出來的物質。可經由與受酶質之交互作用，直接或間接檢測該標記。適當的實例包括但不限於酵素、有色的染料、螢光物質、化學發光物質、生物發光物質和放射性同1素在較佳的具體貫施例中，該可檢測標記爲酵素。 L ^使酵素與對已結合之抗體-抗原複合物具有結合親和力的物質連結。該標記對該複合物的結合作用可藉著抗體_抗原、蛋白質·配體或抗生物素蛋白（鏈黴素抗生物素蛋白卜 13- 本纸張从適用中國國家標準（CNS ) Μ· ( 2數297公着 (請先閱讀背面之注意· 訂562925 V. Description of the invention (10 trays; such as nitrocellulose, fiber boast, succinct fiber, fibrin, bismuth bismuth ester, polycarbonate beads, tubing packing materials, etc. Particles, as well as the binding function of the four main components, ... Pan-antigen makes it attached to the bamboo surface of Yakami. Tongdang non-fisherman-covered antigen is smeared on a solid support and adsorbed on it . In a specific embodiment, the human upper part of the upper guard made the coated antigen adsorbed on the microtiter plate. 0 =: (B) 'in the free antigen, that is, the unknown amount of toenails in the sample, and fixed Between the antigens, that is, between the bound antigens, an y-SS hydrazine (Affinity antibody on the binding site competes for binding sites to form bound and unbound antigen-antibody complexes. The bound complex is separated from the unbound complex. I can use any method known in the art to separate it, such as but not limited to = separation, magnetic separation, and washing to remove unbound Complex. Once in the unbound antigen-antibody complex If it is isolated, then in step (D) ^ a detectable label is used to indicate the bound complex. The label can be any substance that can be fixed on the bound antigen-antibody complex and can be detected. Detect the label directly or indirectly through interaction with the enzyme. Suitable examples include, but are not limited to, enzymes, colored dyes, fluorescent substances, chemiluminescent substances, bioluminescent substances, and radioactive isotopes. In the embodiments, the detectable label is an enzyme. L ^ connects the enzyme to a substance having a binding affinity for the bound antibody-antigen complex. The binding effect of the label on the complex can be achieved through antibody_antigen, protein · Ligand or Avidin (Streptomycin Avidin Bu 13- This paper is from the applicable Chinese National Standard (CNS) M · (Number 2297) (Please read the note on the back first. Order

經濟部中央標準局員工消費合作社印製 562925 A7 B7 五、發明説明（ 11 經濟部中央標準局員工消費合作社印製生物素的結合作用。在較佳的具體實施例中，該結合作用爲抗體-抗原的結合作用，其中使該標記與對已結合之抗， -抗原複合物有結合親和力的抗體結合。這樣的二致典型的此項技藝中已熟知的”三明治”構型。與該標記結合的抗體可以是多株或單株的。此外，也可以使用:有^ 體之結合區域的任何上文詳述之抗體的抗體片段，諸: Fab片段。與該標記連結的特定抗體，當然將依據與抗原連結之抗體的種類。也就是説，與該標記連結的抗體將選擇對固定不動之抗體-抗原複合物有結合親和力者。例如，在較佳的具體實施例中，在兔子體内會使與本發明之抗原連結的抗體昇高。結果，與該標記連結的抗體是對兔子抗體有結合親和力的抗-兔抗體。一旦標示了固定不動的抗體-抗原複合物，便在步驟（£) 中引起可測定之變化。可藉著利用紫外光、螢光、電波形的刺激引起可測定的變化，或是受酶質與該標記進行交互作用的導入，產生可測定的變化。已知該可測定之變化，可以是標記固有的，例如該標記可以是放射性同位素，可單純地藉著導入來誘發其中的放射性，例如r射線，達到測定放射性之目的。在一個具體實施例中，係藉著使已經標示之複合物與受酶質接觸而引起可檢測之變化。這類受酶質是此項技藝中已熟知的，當然也依據所使用之標記的種類。在較佳的具體實旅例中.，該標記爲酵素而該受酶質爲能夠與該酵素交互作用的化合物，產生可測定之變化。在更佳的具體實施 -14 本紙張尺度適用中國國家標準（CNS ) A4規格（21〇χ297公楚 (請先閱讀背面之注意事^^寫本頁} t 、πPrinted by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 562925 A7 B7 V. Description of the Invention (11 The binding effect of the biotin printed by the Consumer Standards Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs. In a preferred embodiment, the binding effect is an antibody- Antigen-binding action, in which the tag is bound to an antibody that has binding affinity to the bound, -antigen complex. Such a two-way typical "sandwich" configuration is well known in the art. Binding to the tag The antibody can be multiple or single. In addition, antibody fragments of any of the antibodies detailed above with binding regions can also be used: Fab fragments. The specific antibody linked to the label, of course, will Depending on the type of antibody linked to the antigen. That is, the antibody linked to the label will choose one that has binding affinity for the immobilized antibody-antigen complex. For example, in a preferred embodiment, in a rabbit The antibody linked to the antigen of the present invention is increased. As a result, the antibody linked to the label is an anti-rabbit antibody having a binding affinity for a rabbit antibody. Once the immobilized antibody-antigen complex is labeled, it causes a measurable change in step (£). The measurable change can be caused by the use of ultraviolet, fluorescent, or electrical wave stimulation, or it can be affected by enzymes. Interaction with the introduction of the label results in a measurable change. It is known that the measurable change can be inherent to the label. For example, the label can be a radioisotope, and the radioactivity can be induced simply by introduction, R-rays achieve the purpose of measuring radioactivity. In a specific embodiment, a detectable change is caused by contacting the labeled complex with an enzyme. Such enzymes are well known in the art. Of course, it also depends on the kind of label used. In a better specific practical example, the label is an enzyme and the substrate is a compound capable of interacting with the enzyme, resulting in a measurable change. The specific implementation of -14 This paper size is applicable to the Chinese National Standard (CNS) A4 specification (21〇χ297 公楚 (please read the notes on the back first ^^ write this page) t, π

經濟部中—IIMinistry of Economic Affairs-II

的抗體-抗原複合物。在步驟(C)中，使利用:二=Antibody-antigen complex. In step (C), make use of: two =

本紙張尺度適用中國圉家標準(CNS 562925This paper size is in accordance with Chinese standards (CNS 562925)

^ ^ ’孩酵素爲磷酸酶且該受酶質爲磷酸化合物。酵素與爻酶質的交互作用，通常使得該化合物具有可測定之特唑:例如，化合物爲螢光的，具有強烈的吸光度等等。旦在步驟（E)中引起可測定之變化，就在步驟 ' 、測疋二醯基肼。這類測定係藉著此項技藝中已熟知的万去，例如紫外光分光光度計、螢光分析、^計數等等。 t知上文詳述的分析通常是間析。#明，直接分析 :二^本發明的範圍内。一開始，直接的方法包括步驟⑷ 均二含未知含量之二醯基肼的試樣。該試樣和二醯基胼叼如同上述。肼樣，在步驟(B)中與固定不動、對二酿基複-; 抗體接觸，形成固定的抗體_二酿基耕俨:。⑽體如同上述，最好是對RH2651會昇高的抗體⑽體固定在具有負責附接這類抗體之表面的固二：』。通當的實例包括但不限於微量滴定盤；諸如狀二“酸醋、聚碳_的膜及具有能夠:合::::上:，羞 …、 < 抗又結合功能部位的衍生定在：、fi佳具體實施例中，該抗體爲塗覆抗體，將其固在上述有關塗覆抗原的固體支撑物上。在步驟（B)中，在固定多妷γ轉原裝滿，A 1 a A 儿肢上的結合位置被自由的抗縣滿纟就疋在試樣中未μ^ ^ 'The enzyme is a phosphatase and the enzyme is a phosphate compound. The interaction of enzymes and peptidases usually makes the compounds measurable: for example, the compounds are fluorescent, have a strong absorbance, and so on. Once a measurable change is caused in step (E), the dihydrazide is measured in step '. This type of measurement is accomplished by methods well known in the art, such as ultraviolet spectrophotometer, fluorescence analysis, fluorescence counting, and the like. It is known that the analysis detailed above is usually interstitial. # 明， Direct analysis: within the scope of the present invention. At the outset, the direct method involves step ⑷, both of which contain an unknown amount of dihydrazide. This sample and difluorenyl group are as described above. Hydrazine-like, in step (B), is contacted with an immobilized, secondary antibody; to form an immobilized antibody_secondary base. The carcass is the same as above. It is best to fix the antibody that the RH2651 will raise to the surface with the surface that is responsible for attaching this antibody: ". Common examples include, but are not limited to, microtiter plates; membranes such as di-acid vinegar, polycarbonate, and those with the ability to ::::: 上:, derivation of anti-binding functional sites are determined at : In the specific embodiment of fi, the antibody is a coated antibody, which is fixed on the solid support related to the above-mentioned coated antigen. In step (B), the immobilized poly γ is transferred to the original full, A 1 a A The joint position on the child's limb was not found in the sample by the free anti-county

經濟部中央標準局員工消費合作社印裝 562925 A7 ---------— B7______ 五、發明説明（13 ) "" 標示的二醯基肼抗原與未結合來自試樣中之苯甲醯胼的固定抗體上的位置接觸。因此已標示之抗原將會裝滿抗體上殘餘的結合位置。因爲固定抗體將會連結已標示和未^示之抗原。該二醞基肼抗原和標記均如同上述。然而，在車六佳的具體實施例中，該抗原爲RH2651而該標記爲酵素，最好是辣根過氧化酶（HRP)。最後’在步驟（D )中引起標記中可測定的變化，並在步自（E)中測定二醯基肼。引起可測定的變化和測定二醯基胼，均如同上文有關間接分析的描述。在本文中描述關於諸如rH5992之類的二醯基肼，及其諸如RH2703、RH2651、RH0345和RH2485之類衍生物的分析，足以應付FIFRA註册所需應用之野外殘餘物研究的敏感性。此外，該分析與不同結構之類似物具有足夠的交叉反應性，像是RH0345 ,咸相信該分析將可適用於全部種類的經取代N-第三-丁基-N，Ν’-苯甲醯胼除害劑。撇開該分析的敏感性（百萬分之一）及其以RH5992爲代表之除害劑類的一般用途不提，最大的優點可能是增加的實驗室效力。例如，已經顯示出一個分析師每天可輕易地處理二十個試樣。這個判斷包括試樣的製備和結果的計算。假設每天1 0小時，每個試樣的工作成本僅負擔大約3 〇人_ 分鐘。 •本發明之抗體的額外用途包括使該抗體與固體支撑物結合’以便濃縮來自試樣基質的殘餘物，而使得可以利用營光標來標示之，並使用於組織研究中，以便確認除害劑 ·16_ 本g尺度適用中國國家標準（CNS )八4胁（210Χ297公釐) ' ""^Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 562925 A7 ------------- B7______ V. Description of the invention (13) " " The labeled dihydrazide antigen and unbound benzene from the sample Formazan contacts on immobilized antibodies. The labeled antigen will therefore fill the remaining binding sites on the antibody. Because immobilized antibodies will bind labeled and unlisted antigens. The dihenylhydrazine antigen and label are as described above. However, in a specific embodiment of Che Liujia, the antigen is RH2651 and the label is an enzyme, preferably horseradish peroxidase (HRP). Finally ' causes a measurable change in the label in step (D), and dihydrazide is measured in step (E). Cause measurable changes and the determination of difluorenylfluorene, as described above for indirect analysis. The analysis of dihydrazide such as rH5992 and its derivatives such as RH2703, RH2651, RH0345, and RH2485 is described in this paper, which is sufficient to cope with the sensitivity of field residue studies for applications required for FIFRA registration. In addition, the analysis is sufficiently cross-reactive with analogues of different structures, such as RH0345. Xian believes that the analysis will be applicable to all kinds of substituted N-third-butyl-N, N'-benzidine胼 Pesticides. Leaving aside the sensitivity (parts per million) of this analysis and its general use of pesticides represented by RH5992, the biggest advantage may be the increased laboratory effectiveness. For example, it has been shown that one analyst can easily process twenty samples per day. This judgment includes the preparation of the sample and the calculation of the results. Assuming 10 hours per day, the working cost of each sample only burdens about 30 people_minutes. • Additional uses of the antibody of the invention include binding the antibody to a solid support so as to concentrate the residue from the sample matrix, so that it can be labeled with a cursor and used in tissue studies to identify pesticides · 16_ This g standard is applicable to China National Standard (CNS) 44 胁 (210 × 297 mm) '" " ^

經濟部中央標準局員工消費合作社印製 562925 五、發明説明（14 ) ί細胞隔間：的位置。該資訊提供了有關特定二酿基耕化 σ物心：用杈式或毒性機制和代謝的重要資訊。再者，可將直接免疫分析方法併入野外八把、斤f外分析測量尺”試驗和測量類型的應用中。在下列的實例及專利説明金沾甘、 j Λ月書的其他邵份中使用下列的縮寫： BCA 二辛可寧酸 BSA 牛血清白蛋白 DCC 二環己基碳化二亞胺 DE Α 二乙醇胺 DMF 二甲基甲醯胺 HRP 辣根過氧化酶 K L Η 鎖孔貝血藍蛋白 N H S Ν -备基3虎拍醯亞胺 P B S 嶙酸緩衝生理食鹽水 PBST P B S 和 0.01 % 吐溫 2 〇 ΡΝΡΡ 對-硝苯基鱗酸鹽實例1 免疫原的合成Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 562925 V. Description of Invention (14) ί Cell compartment: location. This information provides important information on specific secondary brewing-based cultivation σ-physical centers: the use of branch or toxic mechanisms and metabolism. In addition, the direct immunoassay method can be incorporated into the field eight-bar, field-fighting analysis ruler "test and measurement type applications. In the following examples and patent descriptions Jin Zhangan, J Shaoshu and other Shaowen books The following abbreviations are used: BCA dicapryninic acid BSA bovine serum albumin DCC dicyclohexylcarbodiimide DE A diethanolamine DMF dimethylformamide HRP horseradish peroxidase KL 锁 keyhole shell hemocyanin NHS N-Protein 3 Tiger Shoot Imine PBS, Phosphoric Acid Buffered Saline PBST PBS and 0.01% Tween 2 〇ΡΝΡΡ p-Nitrophenylphosphonate Example 1 Immunogen Synthesis

在5毫升梨形的燒瓶中加入2 1微莫耳半抗原，並將其;谷解於200微升DMF(Fisher Scientific )中。在單獨的4 毫升試管中將16毫克DCC(EM Science)和9.1毫克 NHS (Sigma Chemical)溶解於 200 微升的 DMF 中。將DCC 和N H S移至含有半抗原的燒瓶中，並容許在室溫下攪拌該 -17- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210 X 297公釐）A 5 ml pear-shaped flask was charged with 21 micromolar hapten, and lysed in 200 microliters of DMF (Fisher Scientific). In a separate 4 ml tube, 16 mg of DCC (EM Science) and 9.1 mg of NHS (Sigma Chemical) were dissolved in 200 microliters of DMF. DCC and N H S are moved to a hapten-containing flask, and the mixture is allowed to be stirred at room temperature. -17- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm)

562925 A7 B7 五、發明説明（15 ) 反應物大約2小時。將該反應移至冷房中，並在4乇下繼續攪拌過夜。 (請先閲讀背面之注意事562925 A7 B7 5. Description of the invention (15) The reactant is about 2 hours. The reaction was transferred to a cold room and stirring was continued overnight at 4 ° F. (Please read the notes on the back first

在5.4耄升的去離子水中，重新組成载劑蛋白質KLH (Imject® ’ Pierce Chemical Co.，在重新組成時爲 2〇毫克蛋白免在PBS pH 7.2中）。將已經活化之半抗原移至微量離心機（微量離心機235B型，Fisher Scientific)，並離心5分鐘，以移除經取代之尿素沉澱物，將上清液移至重新組成的載劑蛋白質溶液中，並在室溫下繼續攪拌4小時。、11In 5.4 liters of deionized water, the carrier protein KLH was reconstituted (Imject® 'Pierce Chemical Co., when reconstituted, 20 mg of protein was exempt from PBS pH 7.2). The activated hapten was transferred to a microcentrifuge (Microcentrifuge Model 235B, Fisher Scientific) and centrifuged for 5 minutes to remove the substituted urea precipitate, and the supernatant was transferred to the reconstituted carrier protein solution And stir at room temperature for 4 hours. , 11

以14000 rpm離心蛋白質反應混合物5分鐘，以移除沉殿的蛋白質。將全部體積之上清液應用在Swift⑧聚丙烯醯胺脱鹽管柱（Pierce Chemical Co.)上，並以在3毫升溶離份中 0·01 Μ之PBS(pH 7.3)洗脱。在將該試樣完全裝入管柱上之後收集溶離份。得到十份3毫升的溶離份，並在2 8 0毫微米處測足母個落離份的吸光度。收集含有免疫原的溶離份，並藉著BCA法（參見BCA蛋白質分析試劑指示_Pierce Chemical Co ·)測定蛋白質之濃度。經濟部中央標準局員工消費合作社印製如下定出半抗原/蛋白質結合率。建力KLH在〇微克/ 毫升到1200微克/毫升之範圍内，在254毫微米處的吸光度標準曲線。從標準曲線中，將藉著B C A法測定之免疫原溶離份的蛋白質濃度換算成在254毫微米處的吸光度。建立在254毫微米處，在濃度範圍爲RH2703到2 6到260毫微莫耳/毫升，以及RH2651的6.5到2 10毫微莫耳内的半抗原 RH265 1和RH2703之吸光度標準曲線。 •在254毫微米處測得的免疫原之吸光度（Ac<)nj)，減去計算 -18- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐) 562925 經濟部中央標準局員工消費合作社印製 A7 _B7五、發明説明（16 ) 出在254毫微米處由KLH貢獻的吸光度（aklh)，得到因爲半抗原引起之吸光度的昇高（AHap)。從半抗原的標準曲線將該値換算成濃度（毫微莫耳/毫升）。從經過計算之半抗原的濃度（毫微莫耳/毫升）除以測得的免疫原濃度，計算出半抗原與蛋白質之莫耳結合率。 KLH之平均分子量係使用6·7 X 106道爾吞。藉著以670除莫耳結合率’算出每10, 〇〇〇 amu (分子質量單位）KLH的半抗原平均分子數。結果顯示在表2中。實例2 額外免疫原的合成根據實例1之程序來製備免疫原，除了半抗原使用 RH2703之外。結果顯示在表2中。實例3 塗覆抗原的合成根據在實例1中製備免疫原之程序，來製備塗覆抗原 (RH2703 和 RH2651 )，除了以 BSA(Imject⑧，Pierce Chemical Co·)來代替KLH作爲載劑蛋白質之外。在計算莫耳結合率時所使用的BSA之平均分子量，爲68,〇〇〇道爾吞。藉著以6.8除莫耳結合率來計算每10,000 amuBSA的半抗原平均分子數。所製備之塗覆抗原的結果顯示在表2 中。 (請先閲讀背面之注意事Centrifuge the protein reaction mixture at 14000 rpm for 5 minutes to remove the proteins from the sink. The entire volume of the supernatant was applied to a Swift (R) polypropylene desalination column (Pierce Chemical Co.) and eluted with PBS (pH 7.3) at 0.01 M in 3 ml of the fraction. After the sample was completely loaded on the column, the dissolution fraction was collected. Ten 3 ml fractions were obtained, and the absorbance of each individual fraction was measured at 280 nm. The fractions containing the immunogen were collected, and the protein concentration was determined by the BCA method (see BCA Protein Analysis Reagent Instructions_Pierce Chemical Co.). Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs The hapten / protein binding rate is determined as follows. Jianli KLH absorbance standard curve at 254 nm in the range of 0 μg / ml to 1200 μg / ml. From the standard curve, the protein concentration of the immunogen eluate measured by the B CA method was converted into the absorbance at 254 nm. Established absorbance calibration curves at 254 nm at concentrations ranging from RH2703 to 26 to 260 nanomoles / ml and RH2651 to haptens RH265 1 and RH2703 at 6.5 to 2 10 nanomoles. • The absorbance of the immunogen measured at 254 nm (Ac <) nj), minus the calculation -18- This paper size applies to China National Standard (CNS) A4 (210X297 mm) 562925 Employees of the Central Standards Bureau of the Ministry of Economic Affairs A7_B7 printed by the consumer cooperative. V. Description of the invention (16) The absorbance (aklh) contributed by KLH at 254 nm is obtained, and the absorbance increase (AHap) due to the hapten is obtained. This radon was converted to a concentration (nanomoles / ml) from the standard curve of the hapten. The molar ratio of hapten to protein is calculated by dividing the calculated hapten concentration (nanomoles / ml) by the measured immunogen concentration. The average molecular weight of KLH is 6.7 X 106 Dalton. By dividing the molar binding rate by 670 ', the average number of molecules of the half-antigen per 10,000 amu (molecular mass unit) KLH was calculated. The results are shown in Table 2. Example 2 Synthesis of Additional Immunogen An immunogen was prepared according to the procedure of Example 1, except that RH2703 was used for the hapten. The results are shown in Table 2. Example 3 Synthesis of coated antigen The coated antigen (RH2703 and RH2651) was prepared according to the procedure for preparing the immunogen in Example 1, except that BSA (Imject (R), Pierce Chemical Co.) was used instead of KLH as the carrier protein. The average molecular weight of BSA used in the calculation of the molar binding rate was 68,000 Daltons. The average number of molecules of a hapten per 10,000 amuBSA was calculated by dividing the mole binding rate by 6.8. The results of the prepared coated antigens are shown in Table 2. (Please read the notes on the back first

訂Order

___ -19- :紙張从適用中國國家標準（CNS )八4祕（21GX297公釐) 562925 A7___ -19-: Paper from the Chinese National Standards (CNS) Eighty-fourth Secret (21GX297 mm) 562925 A7

562925 A7 B7562925 A7 B7

經濟部中央標準局員工消費合作社印製五、發明説明（18 ) 血液的產製。實例5 兔子抗血清之分析在塗覆緩衝溶液（P B S，pH 7.2 )中重新組成塗覆抗原，至大約1 0微克/毫升之原始濃度。保留9 6孔微量滴定盤中第1和第2行的孔給對照組試樣。開始在第3行的孔中加入 200微升原始濃度的塗覆抗原。從第4到1 2行的孔中，含有 100微升的塗覆緩衝溶液。將一份1〇〇微升、在第3行孔中的塗覆抗原移至第4行的孔中，並與其中的塗覆緩衝溶液混合，並以類似的方式，在每個後續一行的孔中，以塗覆緩衝溶液按1 : 2連續稀釋塗覆抗原。該稀釋過程持續到第 1 1行，其爲1 : 256倍稀釋的塗覆抗原。拋棄最後的1〇〇微升。在第1 2行中的孔僅含有無抗原的塗覆緩衝溶液，作爲陰性對照組。孔A1和A2爲空氣空白，並不接受試劑。孔 B 1和B 2以塗覆杌原塗覆，而成爲背景孔。以i 〇〇微升在塗覆緩衝溶液中之KLH(大約1〇微克/毫升）來塗覆孔Cl* C 2，作爲陽性對照組。剩下的孔D丨到H 2不作處理。將如此製備之培養盤以密封膠帶密封、覆蓋，以塑膠包裝材料包裝，並置於冰箱中在4 °C下培養過夜。在培養過夜之後，從冰箱中移出該培養盤並將其倒空。以300微升BSA封阻溶液（1%BSA，pH72，由溶解於毫升PBS中之！.〇克BSA來製備）來封阻在孔中未結合的結合位置（除了 A 1和A2之外）。在室溫下培養該溶液至少i 〇分鐘。Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (18) Production of blood. Example 5 Analysis of rabbit antiserum The coated antigen was reconstituted in a coating buffer solution (P B S, pH 7.2) to an original concentration of about 10 μg / ml. Save the wells in rows 1 and 2 of the 96-well microtiter plate to the control sample. Begin adding 200 microliters of the original coated antigen to the wells in row 3. The wells in rows 4 to 12 contained 100 µl of the coating buffer solution. A 100 microliter portion of the coated antigen in the well of row 3 was moved to the well of row 4 and mixed with the coating buffer solution therein, and in a similar manner, in each subsequent row of In the wells, the coating antigen was serially diluted 1: 2 with the coating buffer solution. This dilution process continued to line 11, which was a 1: 256 dilution of the coated antigen. Discard the last 100 microliters. The wells in row 12 contained only an antigen-free coating buffer solution as a negative control group. Wells A1 and A2 are blank and do not accept reagents. The holes B 1 and B 2 are coated with the EBARA, and become background holes. 100 μl of KLH (approximately 10 μg / ml) in the coating buffer solution was used to coat the wells as a positive control group. The remaining holes D1 through H2 are left untreated. The culture plate thus prepared was sealed with a sealing tape, covered, and packed in a plastic packaging material, and placed in a refrigerator at 4 ° C for overnight cultivation. After overnight incubation, remove the plate from the refrigerator and empty it. 300 microliters of BSA blocking solution (1% BSA, pH 72, prepared by dissolving in 0.1 ml of PBS! .0 g BSA) to block unbound binding sites in the wells (except A 1 and A2) . Incubate the solution at room temperature for at least 100 minutes.

-21 - 經濟部中央標準局員工消費合作社印製 562925 A7 - -........ - - _ B 7 五、發明説明（19 ) 以封阻溶液按丨：1000來稀釋受試的試樣，並從孔八3開始將200微升應用在a排的孔中。在33到^[3排的孔中含有 100微升的封組溶液。將在A排之孔中的受試試樣（1〇〇微升）和土 B排的孔中’並徹底混合。以類似的方式將試樣連續稀釋1 ·· 2至G3排的孔中（1 : ，並拋棄最後1〇〇微升。Η 3排的孔中不含有抗體，作爲陰性對照組。將背景孔 Β1和Β2與100微升預先放血並以封阻溶液稀釋i : 5〇〇的血清一起培養。將陽性對照組孔C1和C2與1 : 1000倍稀釋之受試試樣一起培養。將培養盤與受試試樣一起培養大約工小時。在培養之後移除受試試樣，並以沖洗緩衝溶液沖洗各孔。以緩衝落液裝滿各孔4 - 5次。在第三或第四次沖洗期間’在拋棄之前容許緩衝溶液停留在各孔中，浸泡3 _ 5分鐘。以1毫升50%的甘油重新組成〇·6毫克、連結有鹼性磷酸酶之山羊抗-兔IgG(Pieixe Chemical Co·)，並以封阻溶液稀釋1 : 5000。將1〇〇微升的該溶液加至每個孔中，並在室溫下培養該培養盤至少丨小時。洗去過量的抗-兔抗體，並在每孔中加入100微升PNPP溶液（5毫克PNPP溶解於8毫升去離子水和2毫升DEA緩衝溶液中）。在室溫下培養該培養盤3 0分鐘。然後加入5 0微升2N NaOH以中止該酵素反應。利用Dynatech MR5000微量滴應盤閱讀機，在41〇毫微米處測定各孔的吸光度。結果描述於表3中。 -22- 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）-21-Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 562925 A7--........--_ B 7 V. Description of the invention (19) Dilute the test solution with the blocking solution by 丨: 1000 Samples, and 200 microliters were applied to the wells in row a starting from well 8-3. The wells in rows 33 to 3 contain 100 µl of the blocking solution. The test sample (100 microliters) in the wells of row A and the wells of row B were mixed thoroughly. The samples were serially diluted in a similar manner to the wells of rows 1 to 2 to G3 (1:, and the last 100 microliters were discarded. Η The wells in row 3 did not contain antibodies and served as negative control groups. The background wells were B1 and B2 were cultured with 100 microliters of pre-bleeded blood and diluted i: 500 with blocking solution. Positive control wells C1 and C2 were cultured with test samples diluted 1: 1000 times. Culture plates Incubate with the test sample for approximately one hour. Remove the test sample after incubation and rinse the wells with a wash buffer solution. Fill each well 4 to 5 times with a buffer solution. At the third or fourth time During the rinse period, allow the buffer solution to stay in each well before discarding, and soak for 3-5 minutes. Reconstitute 0.6 mg of goat anti-rabbit IgG (Pieixe Chemical) with alkaline phosphatase in 1 ml of 50% glycerol Co ·), and diluted 1: 5000 with blocking solution. 100 microliters of this solution was added to each well, and the plate was incubated at room temperature for at least 丨 hours. The excess anti-rabbit was washed away Antibody, and add 100 μl PNPP solution (5 mg PNPP dissolved in 8 ml Water and 2 ml of DEA buffer solution). Incubate the plate for 30 minutes at room temperature. Then add 50 microliters of 2N NaOH to stop the enzyme reaction. Use Dynatech MR5000 microtiter plate reader at 41 mM. The absorbance of each well was measured at a micron. The results are described in Table 3. -22- This paper size applies to China National Standard (CNS) A4 (210X297 mm)

562925 A7 B7 五、發明説明（20 表3 免疫原兔子# 受試血液#1 受試血液敗生產血液#1 生A血液#2 RH2651-KLH 1331 1/32000 1/64000 1/64000 1/128000 ft Μ 1332 1/32000 1/128000 1/64000 1/128000 Μ 11 1333 1/32000 無血液2 1/64000 1/128000 ” ” 1334 1/32000 1/64000 1/128000 1/128000 ft 19 1335 1/32000 1/32000 1/12800.0 1/128000 RH2703-KLH 1336 1/16000 1/64000 1/64000 1/128000 ” 99 1337 1/16000 1/64000 1/64000 1/128000 tt ft 1338 1/16000 1/64000 1/64000 1/128000 ft 9t 1339 1/32000 1/64000 1/64000 1/128000 " M 1340 nd1 1/64000 1/64000 1/128000 未測仔抗體力價。在違動物中耳朵結症防礙採取受試血液。實例6 起ft的分離和站化經濟部中央標準局員工消費合作社印製集中在實例3中的整個注射過程中收集到的免疫血清，並藉著β C A法分析全部的蛋白質，發現爲6 6毫克/毫升。測定免疫血清的體積，並分成各9 〇毫升的兩等份。以兩份體積的去離子水稀釋一等份。接著以一份體積的4 Μ硫酸銨稀釋所得的溶液，得到最後的2 Μ硫酸銨溶液。容許在室溫下攪掉該懸浮液過夜。藉著以iOjOOg-av離心30分鐘收集蛋白質沉澱物。將蛋白質小球溶解於少量的〇.〇2 Μ磷酸鈉緩 —--------23-_ 本紙張又度適用巾國g家標準（⑽）〜胁（21Gx297公楚） 562925 A7 B7 五、發明説明（21 ) 衝溶液（pH 8.0)中，其係由〇·ι Μ磷酸鈉緩衝溶液的稀釋作用來製備之。在室溫下將重新組成的蛋白質溶液對2公升〇·〇2 Μ磷酸鈉緩衝溶液（ρ Η 8 )進行透析四小時，然後在4 °C下對4公升透析過夜，最後在室溫下對4公升透析4小時。得自收集抗血清之IgG的離子交換純化作用，係以兩個獨立的等份，利用DEAE纖維素（DE52， Whatman， Ιη〇·)，濕體積150毫升來經營，將其填塞至2·6公分（内徑）的管柱中，墊高度大約2 6公分。以0.02 Μ磷酸鈉緩衝溶液 (pH 8.0)來平衡該管柱。該管柱具有大約1 5克（每毫升濕體積1 0毫克）的蛋白質容量。將全部體積的透析蛋白質溶液倒入管柱中，並以0.02M磷酸鈉緩衝溶液，pH 8.0開始洗脱。調整流速至大約5毫升/分鐘，並捕捉5毫升溶離份。前15 0毫升的流出物含有任何未保留的蛋白質。在前150毫升·之後，以直線梯度連續改變莫耳濃度至〇·3Μ。在兩個槽中形成梯度，前一個梯度在第一個槽中含有250¾升〇·〇2Μ緩衝溶液’而在第二個槽中含有250毫升〇·3Μ的緩衝溶液。在洗脱作用期間持續攪拌第一個槽。以 5愛：升溶離份爲單位收集下一個500毫升流出物。藉著在 280毫微米處測定吸光度來監視蛋白質之洗脱，將吸光度對洗脱體積作圖（溶離份#)而得到色譜。 •藉#在本文中描述的間接方法來分析每個溶離份之一等份的IgG。收集含有IgG的溶離份，並利用帶有八爪沁抓滤器（分子量排除限制-10,000道爾呑）的揽拌細胞濃縮器減少 (請先閱讀背希之法意事寫本562925 A7 B7 V. Description of the invention (20 Table 3 Immunogen rabbit # Test blood # 1 Test blood fails to produce blood # 1 生 A 血 # 2 RH2651-KLH 1331 1/32000 1/64000 1/64000 1/128000 ft Μ 1332 1/32000 1/128000 1/64000 1/128000 Μ 11 1333 1/32000 bloodless 2 1/64000 1/128000 ”” 1334 1/32000 1/64000 1/128000 1/128000 ft 19 1335 1/32000 1/32000 1 / 12800.0 1/128000 RH2703-KLH 1336 1/16000 1/64000 1/64000 1/128000 ”99 1337 1/16000 1/64000 1/64000 1/128000 tt ft 1338 1/16000 1/64000 1 / 64000 1/128000 ft 9t 1339 1/32000 1/64000 1/64000 1/128000 " M 1340 nd1 1/64000 1/64000 1/128000 Untested antibody titers. Impaired ear disease in animals Test blood was taken. Example 6: Isolation and stationation from ft. The Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs printed the immune serum collected during the entire injection process concentrated in Example 3, and analyzed all proteins by the β CA method. It was found to be 66 mg / ml. The volume of the immune serum was measured and divided into two aliquots of 90 ml each. Diluted with two volumes of deionized water One aliquot. The resulting solution was then diluted with one volume of 4 M ammonium sulfate to give the final 2 M ammonium sulfate solution. The suspension was allowed to stir overnight at room temperature. Collected by centrifugation at iOjOOg-av for 30 minutes Protein precipitate. Dissolve the protein pellets in a small amount of 0.02 M sodium phosphate. -------- 23-_ This paper is again applicable to the national standard (⑽) ~ 胁 (21Gx297) (Ch) 562925 A7 B7 V. Description of the invention (21) In the flushing solution (pH 8.0), it is prepared by the dilution of a sodium phosphate buffer solution of 0 · M. The reconstituted protein solution is prepared at room temperature. Liter 0.02 M sodium phosphate buffer solution (ρ Η 8) was dialyzed for four hours, then 4 liters were dialyzed overnight at 4 ° C, and finally 4 liters were dialyzed at room temperature for 4 hours. Obtained from the collection of antiserum Ion exchange purification of IgG is carried out in two separate aliquots using DEAE cellulose (DE52, Whatman, Ιη〇 ·), a wet volume of 150 ml, and stuffing it to 2.6 cm (inner diameter). In the column, the height of the mat is about 26 cm. The column was equilibrated with a 0.02 M sodium phosphate buffer solution (pH 8.0). The column has a protein capacity of approximately 15 grams (10 mg per milliliter of wet volume). The entire volume of the dialysis protein solution was poured into a column and elution was started with a 0.02 M sodium phosphate buffer solution, pH 8.0. Adjust the flow rate to approximately 5 ml / min and capture 5 ml of dissociated fractions. The first 150 ml of the effluent contained any unretained protein. After the first 150 ml ·, the molar concentration was continuously changed to 0.3 M in a linear gradient. A gradient was formed in two tanks, the first gradient contained 250 ¾ liters of a 2.02 M buffer solution ' in the first tank and 250 ml of a 0.3 m buffer solution in a second tank. The first tank was continuously stirred during the elution. Collect the next 500 ml effluent in 5 liters: liters of fractions. The elution of the protein was monitored by measuring the absorbance at 280 nm, and the absorbance was plotted against the elution volume (dissociation #) to obtain a chromatogram. • Analyze one aliquot of IgG by using the indirect method described in #this article. Collect IgG-containing dissociation fractions and reduce it with a cell concentrator equipped with an Octazone Grain Filter (Molecular Weight Exclusion Limit-10,000 Daur). (Please read the Happiness Book first.

tr 經濟部中央標準局員工消費合作社印製tr Printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs

-24 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐）-24 This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

562925 A7 五、發明説明（22 ) 體積。收集蛋白質的濃縮物，並藉著標準BCa法分析經過濃縮的I g G溶離份。將疊氮化鈉（〇〇2 %重量/體積）加至經過無菌過濾並冷凍儲存在-20。(：下的蛋白質溶液中。得自 DESE離子X換管柱之蛋白質洗脱側面圖描緣於圖j中。空心圓代表全部蛋白質在280毫微米處測得的吸光度。實心圓代表藉著間接分析在410毫微米處測得每個溶離份的抗體力價。收集溶離份5 1 - 8 0，以便藉著蛋白質親和力層析法進行進一步的純化。利用蛋白質A AffinityPak^管柱和ImmunPure® IgG緩衝溶液（ImmunoPure® IgG 純化工具組，Pierce Chemical Co)對經DEAE純化過的I g G ( 1毫克/毫升）進行親和力純化。容許蛋白質A管柱和緩衝溶液回復至室溫，並以5毫升結合緩衝溶液沖洗該管柱。將經過DEAE純化的I g G稀釋1 : 9，得到濃度爲6毫克/毫升的蛋白質，並將1毫升的一等份應用在管柱中。以另1 5毫升的結合緩衝溶液沖洗管柱。以5毫升immunoPure® IgG緩衝溶液完成I g G的洗脱，並補捉1毫升溶離份。在280毫微米處測定每個溶離份的吸光度，並利用已經先以10毫升0.02m PBS(20 mM磷酸鈉， 100 mM NaCl，pH 7.4)調節過的 5 毫升Excellulose® 管柱，將具有明顯吸光度的溶離份（溶離份3 )脱鹽。利用十份1毫升的0.02M PBS洗脱，並收集1毫升溶離份。藉著BCA法分析每個溶離份之吸光度的總蛋白質。將經過蛋白質A親和力層析法純化的I g G稀釋1 ·· 5，得到濃度爲1毫克/毫升的蛋白質。從66毫克/毫升之平均 -25- f請先閲讀背面之注意事562925 A7 5. Description of the invention (22) Volume. The protein concentrate was collected and analyzed for the concentrated Ig G fractions by standard BCa method. Sodium azide (0.02% w / v) was added to sterile filtered and stored frozen at -20. (: In the protein solution below. The side view of the protein elution from the DESE ion X change column is drawn in Figure j. The open circles represent the absorbance of all proteins measured at 280 nm. The solid circles represent the indirect by Analytical antibody titers were measured for each fraction at 410 nm. Fractions 51-80 were collected for further purification by protein affinity chromatography. Protein A AffinityPak ^ column and ImmunPure® IgG Buffer solution (ImmunoPure® IgG purification kit, Pierce Chemical Co) for affinity purification of DEAE-purified I g G (1 mg / ml). Allow protein A column and buffer solution to return to room temperature and use 5 ml The column was rinsed with a buffer solution. The DEAE purified I g G was diluted 1: 9 to obtain a protein with a concentration of 6 mg / ml, and an aliquot of 1 ml was applied to the column. Another 15 ml Wash the column with the binding buffer solution. Complete elution of 1 g with 5 ml of immunoPure® IgG buffer solution and catch up with 1 ml of dissociation. Measure the absorbance of each dissociation at 280 nm. The 5 mL Excellulose® column, which has been conditioned with 10 mL of 0.02m PBS (20 mM sodium phosphate, 100 mM NaCl, pH 7.4), has been desalted to dissolve the fraction (dispersion 3) with significant absorbance. Use ten fractions of 1 1 ml of 0.02M PBS was eluted, and 1 ml of fractions were collected. The total protein of the absorbance of each fraction was analyzed by the BCA method. Ig G purified by protein A affinity chromatography was diluted 1 ·· 5 to obtain Protein at a concentration of 1 mg / ml. Average from 66 mg / ml to -25-f Please read the notes on the back first

經濟部中央標準局員工消費合作社印製本紙張尺度適用中國國家標準（CNS ) A4^洛（210X297公釐） 562925 經濟部中央標準局員工消費合作社印製 A7 ---- B7五、發明説明（23 ) 總血清蛋白質中得到估計爲9毫克/毫升的IgG濃度，IgG 的估計回收値爲6 7 %。實例7 _間接分析的最佳方法利用RH2703 -BSA作爲塗覆抗原。使在最少量抗原 (RH2703 )存在下得到可讀反應（〇 3 _ 〇 5吸光度單位@41〇笑微米）所需的濃度，藉著西洋棋盤式之分析參見v〇Uer 等人 ’ Bull of World Health Organization， 53， 35 (1976)),在固足含量之蛋白質A純化IgG(1()微升的2〇微克/¾升落液，100毫微克/孔）的存在下發揮最大效用。先前已經使蛋白質A純化之IgG的濃度，在固定含量之塗覆抗原的存在下發揮最大效用，得到對最少含量之RH5992的可讀反應。最佳的抗體濃度是大約2 〇微克/毫升（每個微量孔爲1〇〇毫微克），而最佳的塗覆抗原爲25毫微克/毫升或每個微量孔2.5耄微克。當換算成莫耳基礎且假設半抗原/蛋白質結合率爲55時，該半抗原超過所加入之IgG的三_倍莫耳濃度。在最適當之RH2703-BSA塗覆抗原之濃度的存在下，定出 RH2703、RH2651、RH5992、RH0345 和 RH2485 對蛋白質 IgG的抑制百分比。每種受試物質分析七個濃度，範圍從無受試物質之0毫微克/毫升到1〇〇〇毫微克/毫升。每個濃度重複分析七次。提供抗原陰性和抗體陰性孔作爲陰性對照組，同時利用κ L Η /抗κ L· Η孔作爲陽性對照組。 (請先閲讀背面之注意事Printed on the paper of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs. The paper size is applicable to the Chinese National Standard (CNS) A4 ^ Luo (210X297 mm) 562925 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 ---- B7. 23) An estimated IgG concentration of 9 mg / ml was obtained in the total serum protein, and the estimated recovery of IgG was 67%. Example 7-Best Method for Indirect Analysis Using RH2703 -BSA as the coating antigen. The concentration required to obtain a readable response (〇3 _〇5 absorbance unit @ 41〇笑米) in the presence of a minimum amount of antigen (RH2703). See, by checkerboard analysis, v〇Uer et al. 'Bull of World Health Organization, 53, 35 (1976)), which exerts its maximum effect in the presence of a solid content of protein A-purified IgG (1 () microliter of 20 micrograms / ¾ liter of liquid, 100 nanograms / well). The concentration of IgG purified by Protein A has previously been maximized in the presence of a fixed amount of coated antigen, resulting in a readable response to the smallest amount of RH5992. The optimal antibody concentration is approximately 20 μg / ml (100 nanograms per microwell), while the optimal coated antigen is 25 ng / ml or 2.5 μg microwells per microwell. When converted to the Mol basis and assuming a hapten / protein binding ratio of 55, the hapten exceeds the three-fold molar concentration of IgG added. In the presence of the most appropriate concentration of RH2703-BSA coated antigen, the percentage inhibition of protein IgG by RH2703, RH2651, RH5992, RH0345, and RH2485 was determined. Seven concentrations were analyzed for each test substance, ranging from 0 nanograms / ml to 1000 nanograms / ml without the test substance. Each analysis was repeated seven times. Antigen-negative and antibody-negative wells were provided as negative control groups, and κ L Η / anti-κ L · Η holes were used as positive control groups. (Please read the notes on the back first

4 、1Τ4, 1T

-26- 本紙張尺度適用中國國家標準（CNS ) Α4規格（210X297^7 562925 A7 B7 五、發明説明（24 ) 經濟部中央標準局員工消費合作社印製利用含有0· 1 %吐溫2 0的塗覆緩衝溶液進行受試物質母液的連續稀釋，得到1000毫微克/毫升、1〇〇毫微克/毫升、10毫微克/毫升、1毫微克/毫升、ο·ι毫微克/毫升和0.01¾微克/毫升的分析濃度。使用帶有01%吐溫2〇的塗覆緩衝溶液作爲〇毫微克/毫升的分析濃度。在室溫下將受試物質之分析濃度（各190微升）與1 0微升蛋白質a igG —起培養一小時。在培養期間之後，將10q微升受試物質-IgG混合物移至適當的微量滴定盤孔。該微量滴定盤孔已經預先以最佳濃度之RH2703-BSA塗覆過，並以0.3毫升、在塗覆緩衝溶液中的1 % B S A溶液封阻過量的蛋白質結合位置。在室溫下培養該盤額外的一小時。在第二次培養之後沖洗該盤，並加入與鹼性磷酸酶連結的山羊抗-兔免疫球蛋白（〇 6毫克，以1毫升5 〇 %甘油重新組成’且隨後以封阻溶液稀釋i : 5〇00 - Pierce Chmical Co.)。培養該盤，沖洗並按照實例5中之描述加入pNpp受酶質。不加入NaOH中止溶液。並在在410毫微米處讀取吸光度之別，先容許該盤展開兩小時。在圖2到6中描緣抑制曲線。有關純化抗體之特徵的特殊資料顯示在表4中。如同上文的詳述定出π50。藉著與Microsoft Excel© Analysis ToolPak (Microsoft Corporation，Copyright 1993 ) 一致的指數曲線來分析活性百分比對抑制劑濃度的作圖。要计算I C 5〇，解出當y等於5 〇時，也就是殘留5 〇 %活性或 5〇%抑制，指數方程式y = bmnx形式中的乂値。利用 M_S〇ft EXC_，Ver 5 〇的圖式（Microsoft Corporation， (請先閲讀背面之注意事-26- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 ^ 7 562925 A7 B7 V. Description of the invention (24) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs, which contains 0.1% Tween 2 0 The buffer solution was applied to perform serial dilution of the mother substance of the test substance to obtain 1000 nanograms / ml, 100 nanograms / ml, 10 nanograms / ml, 1 nanogram / ml, 0 nanograms / ml, and 0.01¾ Analytical concentration of micrograms / ml. A coating buffer solution with 01% Tween 20 was used as the analytical concentration of 0 nanograms / ml. The analytical concentration of the test substance (190 microliters each) was changed to 1 at room temperature. 0 microliters of protein a igG—incubate for one hour. After the incubation period, transfer 10q microliters of the test substance-IgG mixture to the appropriate microtiter plate wells. The microtiter plate wells have been pre-optimized with RH2703- BSA was coated and blocked excess protein binding sites with 0.3 ml of 1% BSA solution in coating buffer solution. The disc was incubated for an additional hour at room temperature. The disc was rinsed after the second incubation And add with alkaline phosphorus Enzyme-linked goat anti-rabbit immunoglobulin (0.6 mg, reconstituted with 1 ml of 50% glycerol 'and then diluted with blocking solution i: 5000-Pierce Chmical Co.). The dish was cultured, rinsed and The pNpp enzyme was added as described in Example 5. The solution was stopped without the addition of NaOH. The difference in absorbance was read at 410 nm, and the disc was allowed to expand for two hours. The edge inhibition curves are depicted in Figures 2 to 6. Specific information on the characteristics of the purified antibodies is shown in Table 4. π50 was determined as detailed above. The percent activity versus inhibitor was analyzed by an exponential curve consistent with Microsoft Excel © Analysis ToolPak (Microsoft Corporation, Copyright 1993). Plot the concentration. To calculate IC 50, solve when y equals 50, which is the residual 50% activity or 50% inhibition, the exponential equation y = 乂値 in the form of bmnx. Using M_Sft EXC_, Ver 5 〇 diagram (Microsoft Corporation, (Please read the notes on the back first

，1T, 1T

-27- 562925 A7 B7 25 五、發明説明（-27- 562925 A7 B7 25 V. Description of the invention (

Copyright 1993 )完成繪圖。從如同上文詳述的I C 5〇計算交叉反應性的百分比。利用 Microsoft Excel©，Ver 5.0 的圖式（Microsoft Corporation， Copyright 1993 )進行獲自七種抑制劑濃度之七個重複分析之平均吸光度的作圖。可使線性動力範圍具像化，如同從在410毫微米處各種抑制劑濃度之吸光度圖中獲得的曲線之直線部份。如同上述計算敏感性。 _ 決定檢測的範圍爲三倍背景雜音。欲評估背景雜音，計算每個抑制劑濃度之重複分析的標準偏差，並完成回歸分析的最小二乘法。取回歸線的y -截取値作爲因爲雜音引起的背景吸光度。背景吸光度乘以3之因數，並藉著除以由整個直線範圍獲得之標準曲線的斜率換算成抑制劑濃度。將所得的濃度定義爲抑制劑的檢測範圍。 (請先閱讀背面之注意事Copyright 1993) to complete the drawing. The percentage of cross-reactivity was calculated from IC50 as detailed above. The plots of the average absorbance obtained from seven replicate analyses of seven inhibitor concentrations were performed using a chart of Microsoft Excel ©, Ver 5.0 (Microsoft Corporation, Copyright 1993). The linear dynamic range can be visualized as a straight line portion of a curve obtained from absorbance plots of various inhibitor concentrations at 410 nm. Calculate sensitivity as above. _ Determines that the detection range is three times the background noise. To evaluate background noise, calculate the standard deviation of repeated analyses for each inhibitor concentration and complete the least squares method for regression analysis. Take the y-cut of the regression line as the background absorbance due to noise. The background absorbance is multiplied by a factor of 3 and converted to the inhibitor concentration by dividing by the slope of the standard curve obtained from the entire straight line range. The obtained concentration was defined as the detection range of the inhibitor. (Please read the notes on the back first

、1T 經濟部中央標準員· 工消費合作社印製Printed by 1T Central Standards Department, Industrial and Consumer Cooperatives, Ministry of Economic Affairs

表4 受試 IC5〇敏感性檢測範圍交叉反應性物質毫微克/毫升毫微克毫微克/毫升 % RH5992 0.61 0.07 0.66 100 RH2703 1.13 0.19 1.7 54 RH2651 1.59 0.22 1.9 38 RH0345 6.5 0.08 0.75 9 RH2485 0.05 0.08 1.8 NA 實例8 - 1 9Table 4 Test IC50 Sensitivity detection range Cross-reactive substance nanograms / ml Nanograms nanograms / ml% RH5992 0.61 0.07 0.66 100 RH2703 1.13 0.19 1.7 54 RH2651 1.59 0.22 1.9 38 RH0345 6.5 0.08 0.75 9 RH2485 0.05 0.08 1.8 NA Example 8-1 9

28- 本纸張尺度適用中國國家標準（CNS ) Α4規格（210X297公釐）經濟部中央標準局員工消費合作社印製 562925 A7 五7明説明(26 ) " - 間接分析以100微升/孔的RH2703-BSA塗覆抗原（1 0微克/毫升）來塗覆微量滴定盤，得到每孔塗覆1微克。在4下培養該盤過夜。在使用之前先以300微升/孔、在塗覆緩衝溶液中之1 % B S A溶液來封阻未結合的蛋白質結合位置。取得每一等份大約1克的硬花甘藍和土壤試樣。確認硬花甘藍試樣#03 ( 1.0191克）和土壤試樣#49( 1.0180克）爲對照組’並摻入5 0毫微克或500毫微克的R5992，以測量回收。在下文表6中確認試樣身份和試樣質量。知一等份試樣置於25¾升圓錐形離心管中，並將回收試樣摻入50或500毫微克，以PBST稀釋RH5992母液標準物 (1¾克/毫升，在乙腈中）而製備的RH 5992。藉著聲波震盪利用聲波細胞破壞機（Sonicat〇r®，Heat28- This paper size applies to China National Standard (CNS) A4 (210X297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 562925 A7 Five 7 Instructions (26) "-Indirect analysis to 100 microliters / hole RH2703-BSA was coated with antigen (10 μg / ml) to coat a microtiter plate to obtain 1 μg per well. The plate was incubated at 4 overnight. Before use, block unbound protein binding sites with 300 μl / well of 1% B S A solution in a coating buffer solution. Take approximately 1 gram broccoli and soil samples per aliquot. The broccoli sample # 03 (1.0191 g) and the soil sample # 49 (1.0180 g) were confirmed as the control group 'and 50 ng or 500 ng of R5992 was incorporated to measure the recovery. The identity and quality of the samples are confirmed in Table 6 below. Prepare an aliquot of RH prepared in a 25¾ liter conical centrifuge tube, mix the recovered sample with 50 or 500 nanograms, and dilute the RH5992 mother liquor standard (1¾g / ml in acetonitrile) with PBST. 5992. Sonic cell destruction machine (Sonicat〇r®, Heat by Sonic Oscillation)

Systems，Inc. W-225R型，帶有h_1型微形管尖探針），在 8 0%出力下三分鐘以5毫升一等份之PBST萃取試樣。以 4,000g離心該萃取混合物五分鐘，並慢慢倒出pBST層。以 PBST稀釋一等份的各個試樣，得到i : ι〇〇倍稀釋（硬花甘藍試樣）和1 : 500倍稀釋（土壤試樣）的原始試樣萃取物。將一等份經過稀釋之試樣（180微升）與1 〇微升各個 RH5992 標準物（2、10、20、100、200 和 2000 毫微克 / 毫升）和IgG( 10微升）混合，得到具有終rh 5992濃度分別爲 0·1、0·5、1、5、10和100毫微克/毫升的試樣。在以 8 X 1 2排列、9 6孔格式的硼矽酸鹽玻璃管中徹底地混合試樣。相當於抗原陰性孔的管中含有丨〇微升緩衝溶液。相當 _________ -29- 本纸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寫本頁) 訂 562925 A7 B7 五、發明説明（27 於抗體陰性孔的管中不加IgG但含有在ρΒ§τ中i〇〇 ppb的 RH5992。相當於KLH陽性對照組的管子保持空的。在一小時的培養期間之後，將1〇〇微升加料試樣移至微量滴定盤中’並繼續培養另_個小時。在培養該盤之後，如同先前的描述沖洗之，並加入100微升連結有鹼性磷酸酶之山羊抗_兔1§(}，並在室溫下培養一小時。在培養该盤之後再度沖洗，並加入100埤升受酶質。與該受酶質一起培養並持續一到兩個小時，並利用 DynatechMR5000微量培養盤閱讀器，在41〇毫微米處測定吸光度。平均在抗原陰性孔中測得的吸光度，並自動從受試孔中減去。同樣地移除因爲抗體之非-專一性結合而昇高的吸光度。藉著“準加成法來定量在試樣中剩餘的量。利用 Microsoft Excel©，Ver5 〇的圖式，從非專一性結合的平均逆數減去雙重試樣之吸光度，獲得逆吸光度對加入抑制劑之;辰度的作圖。利用 Micr〇s〇ft Excel(g) AnalySis ToolPak，將所得的數據代入y = mx + b的方程式中。在這個關係中，y 是以貫驗測得的逆吸光度，b爲常數，m爲計算出之斜率而 X爲加入之抑制劑的濃度。計算每個數據點相對於得自推測落液之計算値到線性方程式之標準誤差，並從最後的溶液中省略偏離的値。藉著解出設定y等於未加入抑制劑所測得之逆吸光度時的線性方程式之X，獲得試樣的濃度。藉著乘以相對應的稀釋因數將結果換算成總毫微克。藉著將總毫微克除以取得 -30 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） • Ί 請先閲讀背面之注意事Systems, Inc. W-225R, with h_1 type micro-tip probe), extract samples in 5 ml aliquots of PBST for three minutes at 80% output. The extraction mixture was centrifuged at 4,000 g for five minutes, and the pBST layer was decanted. An aliquot of each sample was diluted with PBST to obtain an original sample extract of a 1: 500-fold dilution (broccoli sample) and a 1: 500-fold dilution (soil sample). An aliquot (180 µl) of the diluted sample was mixed with 10 µl of each RH5992 standard (2, 10, 20, 100, 200, and 2000 ng / ml) and IgG (10 µl) to obtain Samples with final rh 5992 concentrations of 0.1, 0.5, 1, 5, 10, and 100 ng / ml, respectively. The samples were thoroughly mixed in borosilicate glass tubes arranged in an 8 X 1 2, 96 well format. The tube corresponding to the antigen-negative well contained 0 microliters of buffer solution. Equivalent _________ -29- This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the note on the back first and write this page) Order 562925 A7 B7 V. Description of the invention (27 for antibody negative wells No IgG was added to the tube but RH5992 containing ioppb in ρΒ§τ. The tube equivalent to the KLH positive control group was left empty. After one hour of incubation, 100 microliters of the feed sample was removed. To the microtiter plate and continue to incubate for another __hours. After incubating the plate, rinse it as described previously, and add 100 μl of goat anti-rabbit __ rabbit1 § ( Incubate for one hour at room temperature. After cultivating the disc, rinse again and add 100 liters of the enzyme substrate. Incubate with the enzyme substrate for one to two hours, and use the DynatechMR5000 microculture disk reader at The absorbance was measured at 0 nm. The absorbance measured in the antigen-negative wells was averaged and automatically subtracted from the test wells. Similarly, the absorbance increased due to the non-specific binding of the antibody was also removed. By "quasi Additive method Remaining amount in the sample. Using Microsoft Excel ©, Ver50 chart, subtract the absorbance of the dual sample from the average inverse number of non-specific binding to obtain a plot of the reverse absorbance versus the inhibitor content. Using Micr0sft Excel (g) AnalySis ToolPak, the obtained data was substituted into the equation of y = mx + b. In this relationship, y is the inverse absorbance measured by a perpetual test, b is a constant, and m is a calculation The slope is given and X is the concentration of the inhibitor added. Calculate the standard error of each data point relative to the calculated 値 obtained from the speculative drop to the linear equation, and omit the 偏离 from the final solution. By solving Set y equal to X of the linear equation when the inverse absorbance was measured without the addition of the inhibitor to obtain the concentration of the sample. The result is converted to total nanograms by multiplying by the corresponding dilution factor. By dividing the total nanograms To get -30 This paper size is applicable to Chinese National Standard (CNS) A4 specification (210X297mm) • Ί Please read the notes on the back first

頁經濟部中央標準局員工消費合作社印製 562925 五、發明説明（28 ) 試樣的質量，計算出最後按毫微克/克計之結杲。建立標準曲線，以便藉著外標準法來定量試樣殘餘物。利用Microsoft Excel© Analysis ToolPak完成指數曲線配合和線性回歸分析。如同上文描述計算回收百分比。藉著分析的外標準法和内標準（加成方法）法兩者，來分析加料5 0毫微克/克和500毫微克/克的回收試樣。關於在括弧中之回收百分比的結果，顯示在表5中。在PBST中和基質空白試樣所獲得的標準曲線之作圖（硬花甘藍試樣 #03和土壤試樣#49)顯示在圖7中。 (請先閱讀背面之注意事Page Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 562925 V. Description of the invention (28) The quality of the sample is calculated as the final result in nanograms / gram. Establish a standard curve to quantify sample residues by external standard methods. Use Microsoft Excel © Analysis ToolPak to complete exponential curve fit and linear regression analysis. Calculate recovery percentage as described above. By using both the external standard method and the internal standard method (addition method), the recovered samples were analyzed by adding 50 ng / g and 500 ng / g. The results of the percentage recovery in parentheses are shown in Table 5. A plot of a standard curve (broccoli sample # 03 and soil sample # 49) obtained by neutralizing a matrix blank sample in PBST is shown in FIG. (Please read the notes on the back first

表5 試樣内標準C毫微克/克外標準C毫微克/克硬花甘藍仏 52(104%) 53(106%) 硬花甘藍r2 471(94%) 426(85%) 土壤Ri 55(110%) 55(110%) 土壤r2 511(102%) 514(103%)Table 5 Internal standard C nanograms / gram External standard C nanograms / gram Broccoli 仏 52 (104%) 53 (106%) Broccoli r2 471 (94%) 426 (85%) Soil Ri 55 ( 110%) 55 (110%) soil r2 511 (102%) 514 (103%)

'1T'1T

經濟部中央標準局員工消費合作社印製得自硬花甘藍和土壤試樣之内標準分析的結果顯示在表6 中。在一週後再分析兩個硬花甘藍和土壤萃取物，以評估試樣基質的穩定性。結果顯示在括弧中。爲每個試樣計算之線性回歸線顯示在圖8到1 5中 -31 - 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） 562925 A7 B7 五、發明説明（29) 表6) 試樣身份 C微克/克試樣身份 C微克/克硬花甘藍土壤 20139-01 1.75(1.41) 92-0017-151 41.4 20139-02 2.41 92-0017-157 20.0 20139-04 5.92 92-0017-175 18.4 20139-05 13.2(12.9) 92-0017-187 - 28 (請先閲讀背面之注意事經濟部中央標準局員工消費合作社印製實例2 0 經標示之抗> 原的合成將4.6毫克（10·5微莫耳）量的RH2651放置在5毫升梨形燒瓶中，並溶解於100毫升DMF中。在分開的試管中，使4.6 毫克NHS和8.8微升DCC各自溶解於100微升的DMF中。將NHS和DCC加至RH265 1中，並在室溫下攪拌大約一小時。在4 °C下持續攪拌過夜。第二天將1 0毫克H RP溶解於 2.7毫升0.01Μ的PBS，pH 7.2中。在Beckman微量離心機 E( Beckman Instruments)中離心該 RH2561 /NHS/DCC 反應混合物三分鐘。在上清液中逐滴加入H RP並攪拌之。在室溫下攪拌該混合物4小時，然後在微量離心機中離心3分鐘。利用以P B S平衡過的Swift®脱鹽管柱（Pierce Chemical Co.)將含有連結蛋白質之上清液脱鹽。以P B S洗脱蛋白質，並收集3毫升溶離份。收集按照在280毫微米處藉著吸光度定出含有蛋白質共軛物的溶離份，並藉著BCA Protein Assay (Pierce Chemical Co.)定出蛋白質之濃度。 -32- 本紙張尺度適用中國國家標準（CNS ) A4規格（210 X 297公釐）The results of standard analysis obtained from broccoli and soil samples printed by the Employees' Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs are shown in Table 6. Two weeks later, two broccoli and soil extracts were analyzed to assess the stability of the sample matrix. The results are shown in parentheses. The linear regression line calculated for each sample is shown in Figures 8 to 15 -31-This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) 562925 A7 B7 V. Description of the invention (29) Table 6) Sample identity C micrograms / gram Sample identity C micrograms / gram Broccoli soil 20139-01 1.75 (1.41) 92-0017-151 41.4 20139-02 2.41 92-0017-157 20.0 20139-04 5.92 92-0017-175 18.4 20139-05 13.2 (12.9) 92-0017-187-28 (Please read the note on the back first. Printed by the Consumer Standards Cooperative of the Central Bureau of Standards of the Ministry of Economic Affairs. 2 0 Labeled Antibodies > The original synthesis will be 4.6 mg (10 5 micromolar) of RH2651 was placed in a 5 ml pear-shaped flask and dissolved in 100 ml of DMF. In separate test tubes, 4.6 mg of NHS and 8.8 µl of DCC were each dissolved in 100 µl of DMF. Add NHS and DCC to RH265 1 and stir for about an hour at room temperature. Continue stirring at 4 ° C overnight. The next day, 10 mg of H RP was dissolved in 2.7 ml of 0.01M PBS, pH 7.2. Centrifuge the RH2561 / NHS / DCC reaction mixture in a Beckman Microcentrifuge E (Beckman Instruments) Three minutes. H RP was added dropwise to the supernatant and stirred. The mixture was stirred at room temperature for 4 hours and then centrifuged in a microcentrifuge for 3 minutes. Using a Swift® desalting column equilibrated with PBS ( Pierce Chemical Co.) desalted the supernatant containing the bound protein. The protein was eluted with PBS and 3 ml of the fractions were collected. The fractions containing the protein conjugate were determined by absorbance at 280 nm. And the protein concentration is determined by BCA Protein Assay (Pierce Chemical Co.) -32- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

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經濟部中央標準局員工消費合作社印裝 562925 Α7 Β7 五、發明説明（30) 如下證實RH2651對HRP的結合作用。以100微升10微克 /毫升2651 -HRP塗覆微量培養盤孔，並儲存在4°C下過夜。在排空該盤後，以1 %在P B S中之B S A封組該孔。在封阻之後，將根據實例6(從1 : 500開始）製備之1 : 2連續稀釋的蛋白質A純化2651 IgG加至該孔中，以探測 RH2651。在室溫下培養卜J、時之後，以〇·01%吐溫2〇 PBS 沖洗該盤。加入以鹼性磷酸酶標示之山羊抗-鳥Ϊ g G，並在室溫下培養額外的1小時。沖洗該盤，並加入對-硝基苯磷阪鹽受酶質。在1 5分鐘的展開時間後，利用Dynatech MR5000微量培養盤閱讀器，在410毫微米處測定吸光度。 RH2651對辣根過氧化酶的結合作用之評估。證實 26；>1IgG對1微克2651-HRP有1 : 32000之力價。這表示一個成功的結合反應。實例2 1 望覆抗體的最佳方法以2651 IgG塗覆微量培養盤。從第1行開始，在每孔中加入100微升的10微克/毫升2651IgG，並越過該盤進行i : 2 之連續稀釋。密封該盤並儲存在4 °C下，直到準備使用爲止。在排空之後，以0.01 %在PBS中之吐溫20沖洗，並在室溫下以在PBS中之1%BSA封阻大約1小時。在封阻之後’排2該盤’並在每孔中加入15 0微升的P B S。然後將 50微升265 1-HRP稀釋液加至培養盤中，以1〇微克/毫升開始，並連續以Γ : 2向下稀釋至培養盤。在室溫下培養i 】時之後’如别述沖洗该盤’並在每孔中加入15 〇微升1 -33- 本紙張尺度適用中國國家標準（CNS ) Μ規格（2i〇x297公釐） '' --— (請先閱讀背面之注意事寫本頁)Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 562925 Α7 Β7 V. Description of the Invention (30) The combination effect of RH2651 on HRP is confirmed as follows. Microwells were coated with 100 μl of 10 μg / ml 2651-HRP and stored at 4 ° C overnight. After emptying the disc, the wells were sealed with 1% B SA in P B S. After blocking, a 1: 2 serially diluted Protein A purified 2651 IgG prepared according to Example 6 (starting at 1: 500) was added to the well to detect RH2651. After incubation at room temperature, the dish was washed with 0.01% Tween 20 PBS. Goat anti-birdfly g G labeled with alkaline phosphatase was added and incubated for an additional hour at room temperature. Rinse the dish and add p-nitrophenylphosphine salt to the enzyme. After a development time of 15 minutes, absorbance was measured at 410 nm using a Dynatech MR5000 microplate reader. Evaluation of the binding effect of RH2651 on horseradish peroxidase. It was confirmed that 26 > 1 IgG has a power value of 1: 32000 for 1 microgram of 2651-HRP. This indicates a successful binding reaction. Example 2 1 Best method for covering antibodies A microplate was coated with 2651 IgG. Starting from line 1, 100 μl of 10 μg / ml 2651 IgG was added to each well, and serial dilutions of i: 2 were made across the plate. Seal the tray and store at 4 ° C until ready to use. After evacuation, rinse with 0.01% Tween 20 in PBS and block with 1% BSA in PBS for approximately 1 hour at room temperature. After blocking, 'row 2 this plate' and add 150 microliters of P B S to each well. Then 50 microliters of 265 1-HRP dilution was added to the culture plate, starting at 10 micrograms / ml, and continuously diluted downward to the culture plate with Γ: 2. Incubate at room temperature] After that, 'wash the disc as stated' and add 150 microliters 1-33 to each well- This paper size applies the Chinese National Standard (CNS) M specification (2i × 297 mm) '' --- (Please read the notes on the back first and write this page)

562925 經濟部中央標準局員工消費合作社印製 Μ Β7______五、發明説明（31 ) Step® Slow-TMB (得自 Pierce Chemical Co.的過氧化酶受酶質）。在展開1小時之後，加入150微升的IN H2S〇4，以中止反應，並利用Dynatech MR5000微量培養盤閱讀器，在 450毫微米處測定每孔的吸光度。對照組包括沒有塗覆抗體的孔，以及其中未加入265 1-HRP的孔。定出0.625微克 /毫升的205 1 IgG塗覆抗體濃度和1.25微克/毫升的2561-HRP濃度，是最適合l-Step® Slow-TMB受酶質使用的濃實例2 2 直接分析以100微升各0.625微克/毫升的2651IgG塗覆微量培養盤的各孔。保留一排不塗覆，作爲對照組。將該盤保持在4 °C下過夜。第二天以在p B S中之0.01 %吐溫2 0沖洗，並以在P B S中之1 %B S A封阻至少3 0分鐘。從1毫克/毫升母液來製造 RH5992、RH2651、RH2703 和 RH2485 的標準溶液。從0.1毫微克/毫升、0.5毫微克/毫升、1毫微克/毫升、5毫微克/毫升、10毫微克/毫升、50毫微克/毫升和100毫微克/毫升，來製備RH0345的標準溶液。在排空封阻溶液之後，在適當的孔中加入150微升的標準物。關於零對照組，加入150微升PBS。在室溫下培養45分鐘之後，在每孔中加入50微升1.25微克/毫升的2651-HRP，並震動培養盤，以便混合内容物。一行含有抗體和標準物的未收到265 1-HRP，但收到50微升？65，作爲陰性對照組。在室溫下培養4 5分鐘之後，如前述沖洗該盤養盤，並在每 ______-34- _ 本紙張尺度適用中國國家標準（CNS ) A4規格（210X297公釐） (請先閲讀背面之注意事 :寫本頁) 訂 4 562925 A7 B7 32 五、發明説明（孔中加入150微升265 1-HRP，再震動該培養盤，以便混合内容物。一行含有抗體和標準物的未收到265 1-HRP，但收到5 0微升P B S，作爲陰性對照組。在室溫下培養4 5分鐘之後’如前述沖洗該盤養盤，並在每孔中加入丨5〇微升Step 1 Slow TMB。在室溫下2小時之後，在每孔中加入15〇微升IN H2S04 ,並利用Dynatech MR5000微量培養盤閲讀器，在45〇毫微米處讀取吸光度。圖16a説明從該分析中產生的典型曲線。見到該曲線的直線部份在0.01毫微克/毫升到1〇毫微克/毫升之間。藉著圖16b解釋在該分析之直線範圍中的典型標準曲線，^ 用逆吸光度產生正的斜率。算出檢測的範圍爲〇()96毫微克 /毫升，並算出敏感性爲0.005毫微克腕州。關於 RH5992和類似物的數據，提供在表7中。表7 (請先閱讀背面之注意事562925 Printed by the Consumer Cooperatives of the Central Bureau of Standards of the Ministry of Economic Affairs Ｍ Β7 ______ V. Description of the Invention (31) Step® Slow-TMB (peroxidase receptor enzyme from Pierce Chemical Co.). After 1 hour of development, 150 µl of IN H2SO4 was added to stop the reaction, and the absorbance of each well was measured at 450 nm using a Dynatech MR5000 microplate reader. The control group included wells without antibody coating, and wells to which 265 1-HRP was not added. Determine the concentration of 205 1 IgG coated antibody at 0.625 μg / ml and the concentration of 2561-HRP at 1.25 μg / ml, which is the most suitable concentration for l-Step® Slow-TMB enzymes. 2 2 Direct analysis at 100 μl Each well of 0.625 micrograms / ml of 2651 IgG coated microplates. One row was left uncoated as a control group. The dish was kept at 4 ° C overnight. Rinse the next day with 0.01% Tween 20 in p B S and block with 1% B S A in P B S for at least 30 minutes. A standard solution of RH5992, RH2651, RH2703 and RH2485 was made from a 1 mg / ml mother liquor. Standard solutions of RH0345 were prepared from 0.1 ng / ml, 0.5 ng / ml, 1 ng / ml, 5 ng / ml, 10 ng / ml, 50 ng / ml and 100 ng / ml. After draining the blocking solution, add 150 μl of standard to the appropriate wells. For the zero control group, 150 µl of PBS was added. After incubation at room temperature for 45 minutes, 50 µl of 1.25 µg / ml of 2651-HRP was added to each well, and the culture plate was shaken to mix the contents. One row contains antibodies and standards. Did not receive 265 1-HRP, but received 50 microliters? 65, as a negative control group. After incubating at room temperature for 4 5 minutes, rinse the discs as described above, and apply the Chinese National Standard (CNS) A4 (210X297 mm) to this paper size (Please read the Note: Write this page) Order 4 562925 A7 B7 32 V. Description of the invention (150 μl of 265 1-HRP is added to the well, and then the culture plate is shaken to mix the contents. One line contains antibodies and standards that were not received 265 1-HRP, but received 50 microliters of PBS as a negative control group. After incubation at room temperature for 4 5 minutes, 'wash the plate as before and add 50 microliters of Step 1 to each well. Slow TMB. After 2 hours at room temperature, add 150 microliters of IN H2S04 to each well and read the absorbance at 45 nanometers using a Dynatech MR5000 microplate reader. Figure 16a illustrates from this analysis Typical curve produced. See that the straight part of the curve is between 0.01 ng / ml to 10 ng / ml. Explain the typical standard curve in the linear range of the analysis by means of Figure 16b. ^ Inverse absorbance Generates a positive slope. Calculated detection range is 0 () 96 millimeters Μg / ml, and calculated the sensitivity to 0.005 nanogram wrist state. Data on RH5992 and the like are provided in Table 7. Table 7 (Please read the notes on the back first

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經濟部中央標準局員工消費合作社印製 IC5〇毫微克/毫升敏感性毫微克檢測範園皇升交叉反應性 RH2651 RH5992Printed by the Consumers' Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs IC50 ng / ml Sensitivity ng Detection Fan Yuan Huang Sheng Cross Reactivity RH2651 RH5992

35- 本纸張尺度適用中國國家標準（CNS ) A4g ( 210X297公釐）35- This paper size applies to Chinese National Standard (CNS) A4g (210X297 mm)

Claims

562925 第086111727號專利申請案 g 中文申請專利範圍修正本（91年10月丨$8六、申請專利範圍562925 Patent Application No. 086111727 g Chinese Patent Application Amendment (October 91 丨 $ 86

1· 一種用於測定二醯棊肼化合物或其衍生物之免疫分析方法之免疫原組合物，包括·· 下式之化合物1. An immunogen composition for an immunoassay method for determining a dihydrazine compound or a derivative thereof, comprising a compound of the following formula

R1R1

其中R為-COOH或-CH2C00H，r1及r2為甲基，及 R及R為氫；與載劑物質結合。 2·根據申請專利範圍第丨項之免疫原組合物，其係作為塗覆抗原。 ' 3·根據申請專利範圍第丨項之免疫原組合物，其中該載劑物質係選自包括蛋白質、多醣'合成的聚合物=共聚物。 4·根據申請專利範圍第丨項之免疫原組合物，其中r為· COOH。 5·根據申請專利範圍第1項之免疫原組合物，其中r為 -CH2COOH。 6· —種根據申請專利範圍第1項之免疫原組合物而提昇的多株抗體。 7· —種根據申請專利範圍第4項之免疫原組合物而提昇的多株抗體。 562925Wherein R is -COOH or -CH2C00H, r1 and r2 are methyl groups, and R and R are hydrogen; combined with a carrier substance. 2. The immunogen composition according to item 丨 of the scope of patent application, which is used as a coating antigen. '3. The immunogen composition according to item 1 of the scope of the patent application, wherein the carrier substance is selected from the group consisting of a protein, a polysaccharide, and a polymer = copolymer. 4. The immunogen composition according to item 丨 of the application, wherein r is COOH. 5. The immunogen composition according to item 1 of the application, wherein r is -CH2COOH. 6. · A plurality of antibodies raised according to the immunogen composition of the first patent application. 7. · A plurality of antibodies raised according to the immunogen composition of the fourth patent application. 562925

8. —種根據申請專利範圍第5項之免疫原組合物而提昇的多株抗體。 9· 一種測定二醯基肼或其衍生物的方法，其包括下列步驟： (A) 提供含有未知含量之二醯基肼的試樣； (B) 使該試樣在固定不動之二醯基肼抗原的存在下，與對二醯基肼抗原具有結合親和力之多株抗體接觸，以致於形成已結合和未結合的抗體·抗原複合物；其中該多株抗體係由包括下式化合物之免疫原所提昇：8. A variety of antibodies raised according to the immunogen composition of the scope of application for item 5. 9. · A method for determining dihydrazine or a derivative thereof, comprising the following steps: (A) providing a sample containing an unknown amount of dihydrazine; (B) placing the sample in a fixed dihydrazide In the presence of a hydrazine antigen, it is contacted with a plurality of strains of antibodies having binding affinity for the dihydrazide antigen, so that a bound and unbound antibody-antigen complex is formed; wherein the multiple strains are immune systems including compounds of the formula The original promotion:

其中R為-COOH或·<：Η2(：ΟΟΗ，R1及為甲基，及 R3及R4為氫；與載劑物質結合； (C)使未結合的抗體·抗原複合物與已結合的抗體-抗原複合物分離； (D )以可檢測之標記來標示該已結合之抗體複合物； (E) 引起該標記中可測定的變化；並 (F) 測定在該試樣中的二醯基肼。 10·根據申請專利範圍第9項之方法’其中該可檢測的標記係選自酵素、有色染料、螢光物質、化學發光物質、生 -2- 本紙張尺度適用中國國家標準(CNS) A4規格(210X 297公釐） 562925 A8 B8 C8Where R is -COOH or <: Η2 (: 〇〇Η, R1 and R are methyl, and R3 and R4 are hydrogen; bound to a carrier substance; (C) unbound antibody-antigen complex and the bound Antibody-antigen complex separation; (D) labeling the bound antibody complex with a detectable label; (E) causing a measurable change in the label; and (F) measuring dioxin in the sample 10. The method according to item 9 of the scope of the patent application 'wherein the detectable label is selected from the group consisting of enzymes, colored dyes, fluorescent substances, chemiluminescent substances, and raw materials.-This paper size applies Chinese national standards (CNS ) A4 size (210X 297 mm) 562925 A8 B8 C8

物發光物質和放射性同位素。 11·根據申請專利範圍第9項之方法，其中該可檢測之標記為能夠與該抗體結合的酵素-抗體共轭物。 12· —種測定二醯基肼或其衍生物的方法，其包括下列步驟： (A) 提供含有未知含量之二醯基肼的試樣； (B) 使該試樣與固定不動且對二醯基肼抗原具有結合親和力之多株抗體接觸，形成固定不動的抗體-抗原複合物； (C) 使、纟可檢測標兄標不之二酿基耕抗原與未複合之固定不動的多株抗體接觸，形成經過標示之抗體-抗原複合物； (D )引起該標記中可測定的變化；並 (E)測定在該試樣中的二醯基肼，其中該固定不動的多株抗體係由包括下式化合物之免疫原所提昇：Luminescent substances and radioisotopes. 11. The method according to item 9 of the scope of patent application, wherein the detectable label is an enzyme-antibody conjugate capable of binding to the antibody. 12. · A method for determining dihydrazide or a derivative thereof, comprising the following steps: (A) providing a sample containing an unknown amount of dihydrazide; (B) immobilizing the sample against the The hydrazine antigen has multiple antibodies with binding affinity to contact to form an immobilized antibody-antigen complex; (C) enables detection and detection of two or more non-complexed immobilized antigens and uncomplexed immobilized multiple strains The antibody contacts to form a labeled antibody-antigen complex; (D) causes a measurable change in the label; and (E) determines the dihydrazine in the sample, where the immobilized multi-strain antibody system Boosted by an immunogen comprising a compound of the formula:

其中R為- COOH或_CH2COOH , Rl及R2為甲基，及 R3及R4為氫；與載劑物質結合。 -3- 本紙張尺度適用中國國家標準(CNS) A4規格(210X297公爱) ^0^925Wherein R is -COOH or _CH2COOH, R1 and R2 are methyl groups, and R3 and R4 are hydrogen; combined with a carrier substance. -3- This paper size applies to China National Standard (CNS) A4 specification (210X297 public love) ^ 0 ^ 925

i3·根料請專_圍第12項之方法，其巾該可檢測之標記係選自酵素、有色染料、螢光物質、化學發光物質、生物發光物質和放射性同位素。 14.根據申請專利範圍第12項之方法，其中該可檢測之標記為能夠與該抗體結合的酵素-抗原共軛物。 15· —種測定二醯基肼的套組，其包括： (A)對二醯基肼有結合親和力的多株抗體，其係由包括下式化合物之免疫原所提昇： R1i3 · Root material, please use the method of item 12 above. The detectable mark is selected from enzymes, colored dyes, fluorescent substances, chemiluminescent substances, bioluminescent substances, and radioisotopes. 14. The method according to item 12 of the application, wherein the detectable label is an enzyme-antigen conjugate capable of binding to the antibody. 15. · A kit for determining dihydrazine, including: (A) multiple strains of antibodies having binding affinity for dihydrazide, which are enhanced by an immunogen including a compound of the formula: R1

其中R為-C00H或-CH2COOH，R1及R2為甲基，及 R3及R4為氫；與載劑物質結合； (B) 能夠與該多株抗體結合的標記；以及 (C) 能夠在該標記的存在下，產生可測定之變化的受質。根據申請專利範圍第1 5項之套組，進一步包括二醯基肼抗原，其包括下式之化合物： -4- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐) 562925 、申請專利範圍 A8 B8 C8 D8Where R is -C00H or -CH2COOH, R1 and R2 are methyl groups, and R3 and R4 are hydrogen; bound to a carrier substance; (B) a label capable of binding to the multiple antibodies; and (C) capable of being labeled at the label In the presence of, it produces measurable changes in substrate. According to the set of 15 items in the scope of patent application, it further includes a dihydrazide antigen, which includes a compound of the following formula: -4- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 562925 、 Application scope: A8 B8 C8 D8

其中R為-COOH或-CH2COOH，R1及R2為甲其 3 4 巧丁丞，及 R及R為氫。 17·根據申請專利範圍第15項之套組，其中該標記為連結至對該多株抗體有結合親和力之抗體的酵素。 18·根據申請專利範圍第15項之套組，其中該標記為連結至對該多株抗體具有結合親和力之二醯基肼抗原的酵素。 19·根據申請專利範圍第9項之方法，其中該固定不動的抗原包括下式之化合物：Wherein R is -COOH or -CH2COOH, R1 and R2 are methyl and trimethylamine, and R and R are hydrogen. 17. The set according to item 15 of the scope of patent application, wherein the label is an enzyme linked to an antibody having a binding affinity for the plurality of antibodies. 18. The kit according to item 15 of the scope of patent application, wherein the label is an enzyme linked to a dihydrazide antigen having binding affinity to the multiple strains of antibodies. 19. The method according to item 9 of the scope of patent application, wherein the immobilized antigen includes a compound of the formula:

^其中R為-COOH或- CH2C00H，Rl及R2為甲基，及 R3及R4為氫；固定至固體支持物。 20·根據申請專利範圍第丨2項之方法，其中該經標記之二醯基胼抗原包括下式之化合物：^ Wherein R is -COOH or -CH2C00H, R1 and R2 are methyl groups, and R3 and R4 are hydrogen; fixed to a solid support. 20. The method according to item 2 of the scope of the patent application, wherein the labeled dimeryl antigen comprises a compound of the formula:

562925 8 8 8 8 A B c D562925 8 8 8 8 A B c D

Chi3 申請專利範圍Chi3 patent application scope

C-NH 其中R為-COOH或-CH2COOH，R1及R2為甲基，及 R3及R4為氫；結合至檢測標記。 6- 本紙張尺度適用中國國家標準(CNS) A4規格(210 X 297公釐)C-NH where R is -COOH or -CH2COOH, R1 and R2 are methyl, and R3 and R4 are hydrogen; bound to a detection label. 6- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)