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 - Google Patents

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 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)

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^ 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

, 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 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 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, 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.

The 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

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 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

Printed 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

Ministry of Economic Affairs-II

Antibody-antigen complex. In step (C), make use of: two =

This paper size is in accordance with Chinese standards (CNS 562925)

^ ^ '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

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) '" " ^

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

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 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

In 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

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

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___ -19-: Paper from the Chinese National Standards (CNS) Eighty-fourth Secret (21GX297 mm) 562925 A7

562925 A7 B7

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-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 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 Printed by the Consumer Cooperatives of the Central Bureau of Standards, Ministry of Economic Affairs

-24 This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

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

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, 1T

-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

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-27- 562925 A7 B7 25 V. Description of the invention (

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

Printed by 1T Central Standards Department, Industrial and Consumer Cooperatives, Ministry of Economic Affairs

Table 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- 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, 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

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

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%)

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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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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 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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Printed 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- This paper size applies to Chinese National Standard (CNS) A4g (210X297 mm)

Claims (1)

562925 Patent Application No. 086111727 g Chinese Patent Application Amendment (October 91 丨 $ 86 1. An immunogen composition for an immunoassay method for determining a dihydrazine compound or a derivative thereof, comprising a compound of the following formula R1 Wherein 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. 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: Where 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 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: Wherein 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 · 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 Where 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 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: ^ 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 D Chi3 patent application scope 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)