JPH10310536A - Immunoassay method - Google Patents

Abstract

(57) [Summary] (Modified) [PROBLEMS] To provide an immunogen, an antibody, a kit and a method for measuring a diacylhydrazine compound using the same. The following formula bonded to a carrier material: Wherein R, R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, (C ₁ -C ₆ ) alkyl and substituted (C ₁
-C _{6) alkyl, (C} 2 -C ₆₎ alkenyl and substituted _(C 2 -C _{6) alkenyl,} a compound of _(C 1 -C _{6) alkoxy, (C} 1 -C ₆₎ alkyloxy and halide) And an antibody produced against the immunogen.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an immunoassay for measuring a diacylhydrazine compound.

[0002] Methods for measuring diacylhydrazine compounds are known. For example, high pressure liquid chromatography (HPLC), gas liquid chromatography (GLC),
There are GC-mass spectroscopy and HPLC-mass spectroscopy, but few are currently available. However, these methods have several disadvantages. These methods generally require expensive and complex equipment. Furthermore, sample preparation and actual analysis are very time consuming and require chemists and technicians specially trained in such methods. Furthermore, such conventional methods do not allow for rapid screening of analogs and / or metabolites in the same step. Rather, it is generally necessary to separate the individual compounds before performing the detection. Therefore, these methods cannot be implemented for fast, low cost measurements, such as those required in field tests or high capacity tests.

[0003] Diacylhydrazine compounds have found particular use as pesticides. Specifically, its use as a caterpillar molting accelerator compound (MAC) has been found. As with all potential toxic chemicals, such pesticides are regulated by the government. A federal insecticide in need of an assay having suitable sensitivity for such use,
Fungicide and rodenticide Act (FIFRA) registration guidelines,
In subdivision O, analysis of residues may be required during the process of obtaining regulatory approval. In addition, the assay has adequate cross-reactivity to the major metabolites of diacylhydrazine rodenticides and is suitable for use in studies of metabolic and environmental effects as required by the FIFRA Registration Guidelines Subdivision N. It must be something. Assays are also required to monitor pesticide use.

[0004] Therefore, there is a need for a diacylhydrazine assay that has sufficient sensitivity and cross-reactivity to meet FIFR guidelines, allows for rapid screening, and is simple and inexpensive to use.

The present inventors have developed immunochemical assays that are sufficiently sensitive and have sufficient cross-reactivity to justify use under the FIFRA guidelines.
In addition, this assay is simple and inexpensive to use.

In a first aspect of the present invention, the following formula bonded to a carrier material:

Embedded image Wherein R, R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, (C ₁ -C ₆ ) alkyl and substituted (C ₁ -C ₆ )
Alkyl, (C ₂ -C ₆ ) alkenyl and substituted (C ₂ -C ₆ )
C ₆ ) alkenyl, (C ₁ -C ₆ ) alkoxy, (C ₁ -C
₆ ) An immunogen is provided which comprises a compound of the formula: alkyloxy and halide.

In a second aspect of the present invention, the following steps are provided: (A) providing a sample containing an unknown amount of diacylhydrazine; (B) providing a sample comprising the presence of an immobilized diacylhydrazine antigen. Contacting with an antibody having a binding affinity for a diacylhydrazine antigen below to form bound and unbound antibody-antigen complexes; (C) unbound antibody -Antibody binding antigen complex-
(D) labeling the bound antibody complex with a detectable label; (E) causing a measurable change in the label; (F) measuring diacylhydrazine in the sample; , Diacylhydrazine or a derivative thereof is provided.

In a third aspect of the present invention, the following steps are provided: (A) providing a sample containing an unknown amount of diacylhydrazine; (B) providing the sample with an immobilized antibody having a binding affinity for a diacylhydrazine antigen. (C) contacting the diacylhydrazine antigen labeled with a detectable label with the uncomplexed immobilized antibody to form a labeled antibody-antigen complex. (D) causing a measurable change in the label; (E) measuring diacylhydrazine in the sample; and a method for measuring diacylhydrazine or a derivative thereof, comprising the steps of:

According to a fourth aspect of the present invention, there are provided the following components: (A) an antibody having a binding affinity for diacylhydrazine; (B) a label capable of binding to the antibody; and (C) a label. A substrate capable of producing a measurable change in the above. A kit for measuring diacylhydrazine is provided.

The term "antigen" as used herein is understood to mean any substance capable of stimulating the production of antibodies. Similarly, the term `` diacylhydrazine antigen '' refers to any diacylhydrazine compound capable of stimulating antibody production, wherein the resulting antibody has binding affinity for diacylhydrazine and derivatives thereof. Understood. Also,
The term "immunogen" is understood to include any substance used to elicit an immune response. Immunogens are generally composed of carrier molecules and other components (haptens).

The term "measurement" as used herein is understood to include both qualitative analysis, ie, confirmation of the presence of an analyte in a sample, and quantitative analysis, ie, determination of the amount of an analyte in a sample. You. It is understood that the term may also include the preparation of standard samples and standard curves well known in the art.

The term "diacylhydrazine compound" as used herein is understood to include, within its scope, diacylhydrazine and metabolites, synthetic analogs, environmental degradation products and photochemical degradation products derived therefrom.

As used herein, the term "IC ₅₀ " refers to the absorbance as one of the absorbances measured in the absence of the inhibitor.
/ 2 is defined as the concentration of inhibitor required to reduce to / 2. This measurement is made by plotting the percentage of residual activity against the concentration of inhibitor. Here, the residual activity ratio (%) is given by the following equation.

Activity (%) = (A ₀ −A _i / A ₀ ) × 100 (where A ₀ is the measured absorbance when no inhibitor is present, and A _i is the measured absorbance at inhibitor concentration i) is there)

Next, inhibition (%) is given by the formula of 100- (activity (%)). Cross-reactivity
reactivity (%) is derived by the following equation: Cross-reactivity (%) = (IC _50,5992 / IC _50, analog) ×
100 (where IC _50,5992 is the IC ₅₀ concentration for RH5992 measured above, and the IC _50, analog is the analog,
That is, I of RH2703, RH2651 and RH0345
_C50 concentration)

Sensitivity in ng is calculated from the inhibitor concentration determined from regression analysis of the standard deviation of a standard concentration repeat analysis as described by the following equation: S = (Y _int / m) × V (where S is sensitivity (ng) and Y _int is Y intercept (unit of absorbance) of regression line from standard deviation of repeated analysis.
Where m is the slope of the regression line (absorbance units per ng / ml) and V is the volume (ml) of the sample applied to the microtiter plate.

The recovery rate (%) is calculated from the following equation. _{% R = (C 0 / C} a) × 100 ( wherein, C ₀ is the observed (measured) concentration, C _a is the actual (spike (spike)) concentration)

As indicated above, immunogens useful for preparing antibodies against diacylhydrazine are provided. Generally, an immunogen comprises a hapten and a carrier molecule.

Haptens are generally diacylhydrazine molecules or derivatives thereof. In one aspect, the hapten is benzoylhydrazine. The benzoyl hydrazine compound is a compound of the above formula (1).

Preferred examples of (C ₁ -C ₆ ) alkyl include methyl, ethyl, n-propyl, isopropyl, n
-Butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl and the like, but are not limited thereto.
Suitable examples of (C ₁ -C ₆ ) substituted alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, substituted by hydroxy, halide, (C ₁ -C ₄ ) alkoxy and nitro. Tert-butyl, n-pentyl, isopentyl, neopentyl, n-
Hexyl and the like, but are not limited thereto.

Preferred examples of (C ₂ -C ₆ ) alkenyl include ethenyl, n-propenyl, isopropenyl, n-
Butenyl, isobutenyl, tert-butenyl, n-pentenyl, isopentenyl, neopentenyl, n-hexenyl and the like, but are not limited thereto. (C
Suitable examples of ₂ -C ₆₎ substituted alkenyl, substituted hydroxy, by a halide or nitro group, ethenyl, n- propenyl, isopropenyl, n- butenyl,
Isobutenyl, tert-butenyl, n-pentenyl,
Examples include, but are not limited to, isopentenyl, neopentenyl, n-hexenyl, and the like.

Preferred examples of (C ₁ -C ₆ ) alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, neopentoxy and n-hexoxy. But not limited thereto.

Preferred examples of (C ₁ -C ₆ ) alkyloxy include carboxy (—COOH), acetyloxy (—CH ₂ COOH), propyloxy (—CH ₂ CH ₂₎
COOH) and n- butyloxy (-CH ₂ CH ₂ CH ₂
COOH), but is not limited thereto.

Preferred examples of the halide include Cl—, B
r-, F- and I-, but are not limited thereto.

In a preferred embodiment, R is --CH ₂ C
OOH, R ¹ and R ² are each methyl,
³ and R ⁴ are each hydrogen. In a more preferred embodiment, R is -COOH, R ¹ and R ² are each methyl, R ³ and R ⁴ are each hydrogen.

The carrier material may be a protein, such as (without limitation), bovine serum albumin, ovalbumin, or keyhole limpet hemocyanin; a polysaccharide, such as (without limitation), dextran, sepharose, agarose or cellulose; or synthetic. It may be a polymer or copolymer, for example (without limitation), polyacrolein, polyamide, polyacrylamide, polybutyrate, polyurea, polyureamide or polystyrene.

In a preferred embodiment, the carrier material is a carrier protein, more preferably bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), most preferably keyhole limpet hemocyanin.

Using the immunogen of the present invention, an antibody against a diacylhydrazine compound can be prepared. The immunogen is introduced into a suitable animal to collect and isolate the antibody,
Purify. The obtained antibody is a polyclonal antibody having high binding affinity and cross-reactivity to diacylhydrazine compounds and derivatives thereof, particularly benzoylhydrazine and derivatives thereof.

Alternatively, a monoclonal antibody can be prepared using an immunogen and a hybridoma method well known in the art.

In one embodiment, the antibodies are raised against an immunogen of formula (1) conjugated to a carrier material.
ised). In a preferred embodiment, the antibody bound to the carrier material, R is -CH ₃ COOH, wherein R ¹ and R ² is methyl, the immunogen of the formula (1) R ³ and R ⁴ are hydrogen To produce. In a more preferred embodiment, the antibody is attached to a carrier material and R is-
It is raised against an immunogen of formula (1) in which COOH, R ¹ and R ² are methyl and R ³ and R ⁴ are hydrogen.

As described above, the present invention provides a method for measuring diacylhydrazine or a derivative thereof.
First, the method includes (A) providing a sample containing an unknown amount of diacylhydrazine.

The sample is generally any material that may contain diacylhydrazine. A preferred example is
Examples include air, water, soil and biological materials. In one aspect, the sample may be an air sample or a sample derived from an air sample. In other embodiments, the sample comprises:
It may be a water sample or a sample derived from a water sample. In yet other embodiments, the sample may be a soil sample or a sample derived from a soil sample.

In one embodiment, the sample may be a biological material or a sample containing or derived from a biological material. Suitable examples of biological material include, without limitation, plant material; blood, serum, plasma, lymph, gastric lavage, bile, vitreous humor
And biological tissues such as plant and animal tissues, and microbiological samples such as bacteria and viruses.

In a preferred embodiment, the sample is soil or plant material or a sample derived therefrom.

The diacyl hydrazine compounds are as described above in formula (1) and derivatives thereof. Such derivatives may be, without limitation, metabolites, synthetic analogs, environmental degradation products, photochemical degradation products derived therefrom. Particularly useful diacylhydrazines are listed in Table 1 below.
Is a benzoylhydrazine of the formula (1) shown below.

[0036]

[Table 1] Benzoyl RR ¹ R ² R ³ R ⁴ Hydrazine RH5992 -CH ₂ CH ₃ -CH ₃ -CH ₃ HH RH2485 H -CH ₃ -CH ₃ -CH ₃ -OCH ₃ RH2703 -CH ₂ COOH -CH _3- CH ₃ HH RH2651 -COOH -CH ₃ -CH ₃ HH RH0345 -Cl HHHH

In step (B), the sample of step (A) is contacted with an antibody having a binding affinity for diacylhydrazine in the presence of an immobilized diacylhydrazine antigen.

The antibody is as described above,
Antibodies produced from immunogens containing RH2651 bound to KLH are preferred.

The antigen of the immobilized diacylhydrazine antigen is
Any diacylhydrazine described above attached to any carrier molecule described above. In a preferred embodiment, the immobilized antigen is RH2651 or RH2703 conjugated to a carrier protein, more preferably RH2703 conjugated to BSA. The combination of antigen and carrier molecule is called a coating antigen
Called.

The coated antigen is generally immobilized on a solid support having a surface that can react sensitively to the attachment of such antigen. Suitable examples include, without limitation, microtiter plates; membrane materials such as nitrocellulose, cellulose, cellulose acetate, polycarbonate, etc .; test tubes; particles such as beads, column packing materials, etc .; Derivatized surfaces having a binding domain capable of binding an antigen are included. Generally, the coated antigen is applied to a solid support and adsorbed thereon.

In one embodiment, the coated antigen is adsorbed on a microtiter plate. In step (B), competition occurs between the free antigen, ie, an unknown amount of benzoylhydrazine in the sample, and the immobilized, ie, bound antigen, for a binding site on an antibody having high affinity for diacylhydrazine. . As a result, bound and unbound antigen-antibody complexes are formed. In step (C), bound complexes are separated from unbound complexes. Separation can be by any means known in the art, including, without limitation, gravity separation, magnetic separation and washing to remove unbound complexes.

Once separated from the unbound antigen-antibody complex, in step (D) the bound complex is
Label with a detectable label. The label can be any material that can be bound to the bound antigen-antibody complex and detected. Labels can be detected directly or indirectly through interaction with a substrate. Suitable examples include, without limitation, enzymes, colored dyes, fluorescent materials, chemiluminescent materials, bioluminescent materials, and radioisotopes. In a preferred embodiment, the detectable label is an enzyme. Generally, the enzyme is bound to a material that has a binding affinity for the bound antibody-antigen complex. Binding of the label to the complex can be by antibody-antigen, protein-ligand, or avidin (streptavidin) -biotin binding. In a preferred embodiment, the binding is an antibody-antigen binding, and the label binds to an antibody having binding affinity for the binding antibody-antigen complex. Such a connection forms a typical "sandwich" structure well known in the art. The antibody conjugated to the label may be polyclonal or monoclonal. Furthermore, Fa
Antibody fragments of any of the antibodies described above that include the antibody binding region, such as the b fragment, can be used. Of course, the specific antibody bound to the label depends on the type of antibody bound to the antigen. That is, the antibody that binds to the label is selected to have binding affinity for the immobilized antibody-antigen complex.

For example, in a preferred embodiment, antibodies bound to the antigen of the present invention are produced in rabbits. Thus, the antibody conjugated to the label is an anti-rabbit antibody having a binding affinity for the rabbit antibody.

Once the immobilized antibody-antigen complex has been labeled, a measurable change is made in the label in step (E). The measurable change can be made by irradiation with UV-visible light, fluorescence and electrical waveforms or by the introduction of a substrate that interacts with the label to produce a measurable change. The measurable change is inherent in the label, for example, the label may be a radioisotope, in which case the activity is, for example, gamma radiation, which can be easily achieved by introducing means for measuring the activity. It is understood that it can be done.

In one embodiment, the measurable change is made by contacting the labeled complex with a substrate. Such substrates are well known in the art and, of course, depend on the type of label used. In a preferred embodiment, the label is an enzyme and the substrate is a compound capable of interacting with the enzyme to produce a measurable change. In a more preferred embodiment, the enzyme is a phosphatase and the substrate is a phosphate compound. The interaction of the enzyme with the substrate generally results in a compound having measurable properties. For example, a compound that is fluorescent has a strong absorbance, and so on.

Finally, once a measurable change has occurred in step (E), the diacylhydrazine is measured in step (F). Such measurements are made by methods known in the art, for example, UV-visible spectrophotometry, fluorometry, gamma counting, and the like.

The assay described above is generally known as an indirect assay. It is also understood that direct assays are included within the scope of the present invention. First,
The direct method involves, in step (A), providing a sample containing an unknown amount of diacylhydrazine. The sample and diacyl hydrazine are as described above.

The sample of step (A) is contacted with an immobilized antibody having a binding affinity for diacylhydrazine in step (B) to form an immobilized antibody-diacylhydrazine complex. Antibodies are as described above, and antibodies raised against RH2651 are preferred.

The antibodies are generally immobilized on a solid support having a surface that can react sensitively to the attachment of such antibodies. Suitable examples include, without limitation, microtiter plates; membrane materials such as nitrocellulose, cellulose, cellulose acetate, polycarbonate, and the like;
Test tubes; particles such as beads, column packing materials, and the like; and derivatized surfaces having binding domains capable of binding antibodies attached thereto. In a preferred embodiment, the antibody is a coated antibody fixed to a solid support as described above for the coated antigen.

In step (B), the binding site of the immobilized polyclonal antibody is filled with free antigen, ie, an unknown amount of benzoylhydrazine in the sample, to form an immobilized antibody-antigen complex. In step (C), a diacylhydrazine antigen labeled with a detectable label is contacted with a site on the immobilized antibody that is not bound to benzoylhydrazine from the sample. Thus, the remaining binding sites on the antibody are filled with the labeled antigen. Thereby, the immobilized antibody binds to the labeled and unlabeled antigen. The diacylhydrazine antigen and the label are
As described above. However, in a preferred embodiment, the antigen is RH2651 and the label is an enzyme,
Preferably, horseradish peroxidase (HR
P).

Finally, in step (D), a measurable change is made in the label and the diacylhydrazine is measured in step (E). Make measurable changes,
The method for measuring diacylhydrazine is as described above for the indirect assay.

RH5992 and its derivative RH2
703, RH2651, RH0345 and RH2485
The assays described herein for diacylhydrazines such as are sensitive enough to apply to the field residue survey required by the FIRA registration. In addition, the assay has sufficient cross-reactivity with structurally dissimilar analogs such as RH0345, ie, the assay has been tested for substituted Nt-butyl-N, N '.
-It is considered applicable to all classes of benzoylhydrazine pesticides.

Apart from the sensitivity (ppb) of the assay to the class of pesticides represented by RH5992 and its general utility, the greatest advantage may be an improved laboratory efficiency. For example, it has been shown that a single analyst can easily process 20 samples per day. This includes time for sample preparation and calculation of results. Assuming 10 hours a day, the operation costs only about 30 persons-per sample.

A further use for the antibodies of the present invention is in binding the antibodies to a solid support, concentrating the residue from the sample matrix, labeling with a fluorescent tag, and removing rodenticide residues in cells. That it can be used in tissue studies to confirm its location.
This information provides important information regarding the mode of action or toxicity and metabolic mechanisms of a particular diacylhydrazine compound. In addition, the direct immunoassay method
Survey typ for field analysis and application
es) dip-stick test
t).

The following abbreviations are used in the following examples and elsewhere in the specification. BCA: bicinchoninic acid BSA: bovine serum albumin DCC: dicyclohexylcarbodiimide DEA: diethanolamine DMF: dimethylformamide HRP: horseradish peroxidase KLH: keyhole limpet hemocyanin NHS: N-hydroxysuccinimide PBS: phosphate buffered saline PBS PBS and 0.01% Tween 20 PNPP: para-nitrophenyl phosphate

Example 1 Synthesis of Immunogen To a 5 ml pear-shaped flask was added 21 μmol of hapten RH2651 and 200 μl of DMF (Fish).
er Scientific). In another 4 ml test tube, 16 mg of DCC (EM Science)
And 9.1 mg of NHS (Sigma Chemical) in 200
Dissolved in μl DMF. DCC and NHS were transferred to the flask containing the hapten and the reaction was stirred at room temperature for about 2 hours. The reaction was transferred to a cooling room and stirring was continued at 4 ° C. overnight.

The carrier protein KLH (Imjec
t, Pierce Chemical Co. , 20m protein in PBS
g) (reconstituted at pH 7.2) was reconstituted in 5.4 ml of deionized water. The activated hapten is transferred to a microcentrifuge (Microcentrifuge Model 235B, Fi
sher Scientific) and centrifuged for 5 minutes to remove displaced urea precipitate. The supernatant was transferred to the returned carrier protein solution and stirring was continued at room temperature for 4 hours.

The protein reaction mixture was prepared at 14000 rpm
The precipitated protein was removed by centrifugation at 5 m for 5 minutes. The entire volume of the supernatant was applied to a Swift polyacrylamide desalting column (Pierce Chemical Co.) and eluted with 0.01M PBS (pH 7.3) in 3 ml fractions. After the sample was completely loaded on the column, fractions were collected. Ten 3 ml fractions were collected and the absorbance at 280 nm was measured for each fraction. Fractions containing the immunogen are pooled and subjected to the BCA method (see BCA protein assay reagent instructions: Pierce Chemical Co.)
The protein concentration was measured by

The hapten / protein binding ratio was determined as follows. A standard curve of KLH absorbance at 254 nm ranging from 0 μg / ml to 1200 μg / ml was constructed. The protein concentration of the immunogen fraction measured by the BCA method was converted from the standard curve to the absorbance at 254 nm. Hapten RH at 254 nm
The standard curves of the absorbances of 2651 and R2703 were measured using RH2
26-260 nmol / ml for 703, RH2
651 was made over a concentration range of 6.5 to 210 nanomoles / ml.

The absorbance of the immunogen was measured at 254 nm (A _conj ) and the KLH calculated contribution to the absorbance at 254 nm.
(A _KLH ) was subtracted to obtain the absorbance (A _Hap ) obtained from the hapten. This value A _Hap was converted from a standard curve for haptens to a concentration (nanomoles / ml).

The molar binding ratio of hapten to protein was calculated by dividing the calculated concentration of hapten (nanomoles / ml) by the measured concentration of immunogen. An average molecular weight of 6.7 × 10 ⁶ daltons for KLH was used. By dividing the molar binding ratio by 670, KLH 10,000,
The average number of hapten molecules per 0 amu was calculated. Table 2 shows the results.

Example 2 Synthesis of a further immunogen An immunogen was prepared according to the procedure of Example 1 except that the hapten used was RH2703. Table 2 shows the results.

Example 3 Synthesis of Coated Antigen KLH was used as a carrier protein in BSA (Imject, Pi
erce Chemical Co. ) Except that the coated antigen (RH2703) was used according to the procedure for preparing the immunogen in Example 1.
And RH2651) were prepared. The average molecular weight of BSA used in calculating the molar binding ratio was 68,000 daltons. By dividing the molar bond ratio by 6.8, B
The average number of hapten molecules per 10,000 amu of SA was calculated. Table 2 shows the results for the prepared coated antigens.

[0064]

Table 2 Sample Protein Hapten Hapten / N ⁽¹⁾ micromol / ml micromol / ml protein RH2651-KLH 2.8 × 10 ^-4 0.437 1560 2 RH2703-KLH 2.9 × 10 ^-4 0.943 3250 5 RH2651-BSA 0.03 1.5 50 7 RH2703-BSA 0.04 2.2 55 8 ⁽¹⁾ N is the number of hapten molecules per 10,000 amu of protein.

Example 4 Preparation of Antibodies 2.5 ml of immunogen (product of Example 2 reconstituted to 1 mg / ml in 0.01 M PBS), 25 mg of M.D. t
Primary inoculum was prepared from uberculosis suspension and 2.5 ml Freund's adjuvant. The mixture was homogenized until it was thick and creamy.

Antibodies were produced using New Zealand white rabbits (5). Rabbits were disease-free female rabbits weighing between 4.75 and 5.5 pounds. Rabbits were first prepared with Bordetella pertussis (B. per, prepared to contain 6 × 10 ¹⁰ cells suspended in 0.6 ml saline).
tussis inoculum), and blood was collected to obtain background titers.

The dose of the primary inoculum per rabbit was 1.0 ml by subcutaneous injection. Inoculate rabbits,
Booster immunizations were received every 21 days. Eight days after the fourth booster injection, rabbits were bled for antibody collection. After a further time, the booster injection was started again and the product was bled again after the fourth booster injection.

Example 5 Assay of Rabbit Antiserum Coated antigen was reconstituted in coating buffer (PBS, pH 7.2) to give a starting concentrate of about 10 μg / ml. 96
The wells of columns 1 and 2 of the well microtiter plate were used for control samples. Starting from the wells in column 3, 200 μl of the coated antigen was added to the starting concentrate.
The coating buffer 10 was added to the wells in columns 4 to 12.
0 μl was included. A portion (100 μl) of the coated antigen in the wells of column 3 was transferred and mixed with the coating buffer in the wells of column 4 and in this way the coated antigen was serially diluted 1: 2 with coating buffer, Placed in the next column well. The dilution process was performed with 1: 2 of the coated antigen.
Continued to column 11, which was a 56 dilution. Last 100μ
l was discarded. The wells in column 12 contained only the coating buffer and no antigen as a negative control sample. Wells A1 and A2 were air blanks and did not contain reagents. Wells B1 and B2
Coated with the coated antigen, which was a background well. Wells C1 and C2 contain 100 μL of KLH (about 10 μg / ml) in coating buffer
l. The remaining wells, D1-H2, were untreated. The plate thus prepared was sealed with sealing tape, covered, covered with plastic wrap, placed in a refrigerator and incubated overnight at 4 ° C.

After overnight incubation, the plates were removed from the refrigerator and transferred to another container. Unbound binding sites in the wells (excluding A1 and A2) were treated with BSA blocking solution (1% BSA, pH 7.2, prepared from 1.0 g BSA dissolved in 100 ml PBS) 3
Blocked with 00 μl. The solution was incubated at room temperature for at least 10 minutes.

The test sample was diluted 1:10 with the blocking solution.
And diluted 200 μl to the wells in row A starting with well A3. Wells in rows B3 to H3 are 100
It contained μl of blocking solution. The test sample (100 μl) in the well in row A was transferred to the well in row B and mixed well. In this way, the samples were placed in G3 row wells (1: 6
4000) 1: 2 serial dilution until the final 100 μl
Was discarded. Wells in row H3 contained no antibody and were negative control samples. Background wells B1 and B2 were incubated with 100 μl of pre-bleed serum diluted 1: 500 in blocking solution. Positive control sample wells C1 and C2 were incubated with a 1: 1000 dilution of the test sample. The plate was incubated with the test sample for about 1 hour.

After incubation, the test sample was removed and the wells were washed with a washing buffer. Wells were filled 4-5 times with buffer. During the third to fourth washes, the buffer was immersed in the wells for 3-5 minutes before discarding. Alkaline phosphatase (Pierce Chemi
cal Co. Goat anti-rabbit IgG conjugated to
g was reconstituted in 50% glycerol and diluted 1: 5000 in blocking solution. 100 μl of this solution is added to each well and the plate is incubated at room temperature for at least 1 hour.
Incubated for hours. The excess anti-rabbit antibody is washed away and 100 μl of PNPP solution (5 mg of PNPP dissolved in 8 ml of deionized water and 2 ml of DEA buffer)
1 was added to each well. Plate at room temperature for 30
Incubated for minutes. Next, 2N-NaOH 50
The enzyme reaction was stopped by adding μl. Dynatech
The absorbance in each well at 410 nm was measured using an MR5000 microtiter plate reader. Table 3 shows the results.

[0072]

[Table 3] Immunogen Rabbit # Test bleed Test bleed Product bleed Product bleed # 1 # 2 # 1 # 2 RH2651-KLH 1331 1/32000 1/64000 1/64000 1/128000 RH2651-KLH 1332 1/32000 1 / 128000 1/64000 1/128000 RH2651-KLH 1333 1/32000 No blood sampling ⁽²⁾ 1/64000 1/128000 RH2651-KLH 1334 1/32000 1/64000 1/128000 1/128000 RH2651-KLH 1335 1/32000 1 / 32000 1/128000 1/128000 RH2703-KLH 1336 1/16000 1/64000 1/64000 1/128000 RH2703-KLH 1337 1/16000 1/64000 1/64000 1/128000 RH2703-KLH 1338 1/16000 1/64000 1/64000 1/128000 RH2703-KLH 1339 1/32000 1/64000 1/64000 1/128000 RH2703-KLH 1340 nd ⁽¹⁾ 1/64000 1/64000 1/128000 (1) Antibody titer was not measured (2) Ear scratches hindered test blood collection in this animal

Example 6 Isolation and Purification of Antibodies Immune sera collected during the course of injection in Example 3 were pooled and assayed for total protein by the BCA method and found to be 66 mg / ml. The volume of the immune serum was measured and divided into two equal aliquots of 90 ml each. One aliquot was diluted with two volumes of deionized water. The resulting solution volume was subsequently diluted with an equal volume of 4M ammonium sulfate to give a final 2M ammonium sulfate solution. The suspension was stirred overnight at room temperature. The protein precipitate was collected by centrifugation at 10,000 g-av for 30 minutes. The protein pellet was dissolved in a minimum volume of 0.02 M sodium phosphate buffer (pH 8.0) prepared from a dilution of 0.1 M sodium phosphate buffer.

The returned protein solution was added to 2 liters of 0.02 M sodium phosphate buffer at room temperature for 4 hours, and then at 4 ° C. for 4 liters overnight.
Finally, dialysis was performed at room temperature against 4 liters for 4 hours.

About 26 cm in a 2.6 cm (id) column
Ion exchange purification of IgG from pooled antiserum was performed using 150 ml of DEAE cellulose (DE52, Whatman, Inc.) with a wet volume of 150 ml packed at bed height. The column was equilibrated in a 0.02 M sodium phosphate buffer (pH 8.0).
The column weighs approximately 1.5 g (10 m / ml wet volume).
g). The entire volume of dialyzed protein solution is pumped over the column and
Elution was started with 2M sodium phosphate buffer, pH 8.0. Adjust the flow rate to about 5ml / min, 5m
One fraction was collected. The first 150 ml eluate contained all unfixed proteins.

After the first 150 ml, the molarity was continuously changed to 0.3 M with a linear gradient. The gradient was formed in a two-chamber gradient former containing 250 ml of 0.02 M buffer in the first chamber and 250 ml of 0.3 ml buffer in the second chamber. The first chamber was continuously stirred during the elution. The next 500 ml of eluate was collected in 5 ml fractions. Protein elution was monitored by measuring absorbance at 280 nm and plotting this against elution volume (fraction #) to obtain a chromatogram.

An aliquot of each fraction was assayed for IgG by the indirect method described here. The fractions containing the IgG were pooled and Amic
The volume was reduced using a stirred cell concentrator equipped with an on filter (molecular weight exclusion limit: 10,000 daltons). The protein concentrate of the pooled and concentrated IgG fraction was assayed by the standard BCA method. Add sodium azide to the protein solution (0.02% w /
v), sterile-filtered and stored frozen at -20 ° C. DEAE
FIG. 1 shows the elution curve of the protein from the ion exchange column. The open circle symbol indicates the total protein measured from the absorbance at 280 nm. Closed circle symbols indicate antibody titers for each fraction measured by indirect assay at 410 nm. For further purification by protein affinity chromatography, fraction 51
Pooled ~ 80.

Protein-A AffinityPak column and I
mmunPure IgG buffer (ImmunoPure IgG purification kit,
Pierce Chemical Co. ) Using DEAE purified IgG
(1 mg / ml) was affinity purified. The Protein-A column and buffer were brought to room temperature and the column was washed with 5 ml binding buffer. DEAE purified IgG was diluted 1: 9 to obtain a protein concentration of 6 mg / ml and a 1 ml aliquot was applied to the column. The column was washed with another 15 ml binding buffer.

IgG was eluted using 5 ml of ImmunoPure IgG elution buffer, and 1 ml of a fraction was collected. The absorbance of each fraction was measured at 280 nm and 0.02M PBS
(20 mM sodium phosphate, 100 mM NaCl,
The fraction having a large absorbance (fraction 3) was desalted using 5 ml of an Excellulose column pre-conditioned with 10 ml of pH 7.4). 1 ml fractions were collected using ten 1 ml aliquots of 0.02 M-PBS for elution. The absorbance of each fraction was assayed for total protein by the BCA method.

The IgG purified by protein-A affinity chromatography was diluted 1: 5 to give a protein concentration of 1 mg / ml. From an average total serum protein of 66 mg / ml, an IgG concentration of 9 mg / ml was estimated,
The estimated recovery of gG was 67%.

Example 7 Optimization of Indirect Assay RH2703-BSA was used as coated antigen. The concentration required to give a readable response (0.3-0.5 absorbance units at 410 nm) in the presence of a minimal amount of antigen (RH2703) is determined by a checkerboard type assay (Voller et al., Bull of World Health). Organizatio
n, 53, 35 (1976)), a fixed amount of protein A purified IgG (10 μl of a 20 μg / ml solution, 10 μl
0 ng / well). Protein A
The concentration of purified IgG was adjusted to a fixed amount of the coated antigen (RH2703).
-BSA, 100 μl of a 10 μg / ml solution) to obtain a readable response to a minimal amount of RH5992.

The antibody concentration was optimized to about 20 μg / ml (100 ng per microwell) and the coated antigen
Optimized to 25 ng / ml or 2.5 ng per microwell. Converted to molar basis and assuming a hapten / protein binding ratio of 55, the hapten is
There is a 3-fold molar excess of G.

RH2703, RH2651 and RH599
2, the% inhibition for RH0345 and RH2485,
Measured against protein IgG in the presence of optimized concentration of RH2703-BSA coated antigen. Each test substance was converted from 0 ng / ml to 1000 in the absence of test substance.
Assays were performed at seven concentrations ranging from ng / ml. The assay at each concentration was repeated seven times. Antigen negative and antibody negative wells provided negative control samples, while KLH / anti-KLH wells were used as positive control samples.

Serial dilutions of the test substance stock solution were performed using a coating buffer containing 0.1% Tween 20 to give 1000 ng / ml, 100 ng / ml and 10 ng.
/ Ml, 1 ng / ml, 0.1 ng / ml and 0.01
An assay concentration of ng / ml was obtained. Coating buffer w /
0.1% Tween 20 was used as 0 ng / ml assay concentration. Test substance assay concentrate (1 each)
90 μl) with protein A IgG (20 μl / m
1) and incubated for 1 hour at room temperature. After the incubation period, the test substance-IgG mixture 100
The μl was transferred to an appropriate microtiter well. Microtiter wells were prepared at the optimized concentration of RH2703-B.
Pre-coated with SA, excess protein binding sites were blocked with 0.3 ml of 1% BSA solution in coating buffer. Plates were incubated for an additional hour at room temperature.

After the second incubation, the plates were washed and alkaline phosphatase (0.6 mg, 5 mg
Returned with 1 ml of 0% glycerol and subsequently diluted 1: 5000 with blocking solution-Pierce Chem
ical Co. ) Was added to the conjugated goat anti-rabbit immunoglobulin. Incubate the plate, wash,
The PNPP substrate was added as described in Example 5. N
Do not add aOH stop solution and proceed in plate for 2 hours (de
After velop) the absorbance at 410 nm was read. The suppression curves are shown in FIGS. Specific data on purified antibody properties is shown in Table 4.

The IC ₅₀ was measured as described above.
A plot of% activity versus inhibitor concentration was generated using Microsoft Excel An
alysis ToolPak (Microsoft Corporation, copyright 19
1993) by exponential curve optimization. Next, I
In order to calculate the C _50, y = a bm ⁿ x of morphology index equation of, y is equal to 50, i.e. 50% active residue or 50%
Solved for x with suppression set. Microsoft Ex
cel, version 5.0 graphics (Microsoft Cor
poration, copyright 1993).

The% cross-reactivity was determined by measuring the IC ₅₀ as described above.
It was calculated from the value. Microsoft Excel, version 5.0
Graphics (Microsoft Corporation, Copyright 19
93) using seven concentrations of inhibitors
A plot of the average absorbance of two replicates was made. The linear variation range was visualized as a linear part of the curve obtained from a plot of absorbance at 410 nm from various inhibitor concentrations. Sensitivity was calculated as described above.

The detection limit was defined as three times the measured background noise. To assess background noise, the standard deviation of the replicates at each inhibitor concentration was calculated and the least squares method of regression analysis was performed.
The y-intercept of the regression line was taken as the background absorbance obtained from the noise. Background absorbance was multiplied by a factor of 3 and converted to inhibitor concentration divided by the slope of the standard curve obtained over the linear range. The resulting concentration was defined as the limit of detection of the inhibitor.

[0089]

Table 4 Test substance IC ₅₀ sensitive detection limits crossreactivity ng / ml ng ng / 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

Examples 8-19 Indirect Assay Microtiter plates were prepared with 100 μl / well of R
Coating with H2703-BSA coated antigen (10 μg / ml) gave 1 μg of coating per well. Plate 4
Incubated overnight at ° C. After blocking unbound protein binding sites, 1% BS in coating buffer
A solution was used with 300 μl / well.

Approximately 1 g aliquots of broccoli and soil samples were taken. Broccoli sample # 3 (1.0191
g) and soil sample # 49 (1.0180 g) as control samples, 50 ng or 500 ng of RH5992 was added, and the recovery was measured. The identification of the sample used and the sample mass are shown in Table 6 below.

A sample aliquot was placed in a 25 ml conical centrifuge tube and the recovered sample was added to PBST diluted R
H5992 stock solution (1 mg / ml in acetonitrile)
50ng or 500ng of RH5992 prepared from was added.

The sample was placed in an ultrasonic cell processor (Sonicator, H
eat Systems, Inc. Model W-225R, Model H-1
A 5 ml aliquot of PBST was extracted by sonication for 3 minutes at 80% power using a microtip probe). Extraction mixture at 4,000 g for 5
After centrifugation for 10 minutes, the PBST layer was decanted. An aliquot of each sample was diluted with PBST to give a 1: 100 dilution of the original sample extract (broccoli sample) and 1: 5
A 00 dilution (soil sample) was obtained.

An aliquot (180 μl) of the diluted sample was
RH5992 standard sample (2, 10, 20, 100, 20
0 and 2000 ng / ml) and IgG (10 μl), respectively, and 0.1,
0.5, 1, 5, 10, and 100 ng / ml final RH
A sample having a 5992 concentration was obtained. The samples were mixed thoroughly in borosilicate glass tubes arranged in a 12 × 8 96-well format. The tube corresponding to the antigen negative well contained 10 μl of buffer. To the tube corresponding to the antibody negative well, add no IgG and add 100p in PBST.
pb of RH5992. The tube corresponding to the KLH positive control sample was left empty.

After a one hour incubation period, 100 μl of the spiked sample was transferred to a microtiter plate and the incubation continued for another hour.

After incubation, the plates were washed as described above, 100 μl of goat anti-rabbit IgG conjugated to alkaline phosphatase was added and incubated for 1 hour at room temperature. After incubation, the plates were washed again and 100 μl of PNPP substrate was added. Incubation with the substrate is continued for 1-2 hours, Dynat
Absorbance was measured at 410 nm using an ech MR5000 microplate reader. The absorbance measured in antigen negative wells was averaged and automatically subtracted from test wells. In this way, the absorbance obtained from binding other than the specific binding of the antibody was removed.

[0097] The residue level in the sample was quantified by the standard addition method. Using Microsoft Excel, version 5.0 graphics, a plot of the reciprocal of absorbance versus the concentration of inhibitor added was obtained from the reciprocal of the average absorbance minus the absorbance from non-specific bindings of the same sample. Microsoft Excel Analysis T
oolPak was used to fit equations of the form y = mx + b. In this relationship, y is the reciprocal of the experimentally measured absorbance, b is a constant, m is the calculated slope, and x is the concentration of the added inhibitor. The standard error of each data point relative to the calculated value from the solution estimated for the linear equation was calculated, and outlying values were excluded from the final solution.

Sample concentrations were obtained by solving a linear equation for x where y was set equal to the reciprocal of the absorbance measured without the addition of the inhibitor. The results were converted to total ng by multiplying by the corresponding dilution factor. By dividing the total ng by the mass of the sample taken,
The final result in ng / g was calculated.

External standard method
Generated a standard curve for quantification of sample residues. Exponential curve optimization and linear regression analysis were performed using Microsoft Excel Analysis ToolPak. % Recovery was calculated as described above.

The recovered samples spiked at 50 ng / g and 500 ng / g were analyzed by external standard method and internal standard method (addition method) analysis. In Table 5,% recovery is shown in parentheses. PBST and matrix blank sample (broccoli sample # 03 and soil sample # 49)
The plot of the standard curve obtained in is shown in FIG.

[0101]

Table 5 Sample Internal standard C ng / g External standard C ng / g Broccoli R ₁ 52 (104%) 53 (106%) Broccoli R ₂ 471 (94%) 426 (85%) Soil R ₁ 55 (110% ) 55 (110%) Soil R ₂ 511 (102%) 514 (103%)

The results from internal standard analysis of broccoli and soil samples are shown in Table 6. The two broccoli and soil extracts were re-analyzed one week later to assess the stability of the sample matrix. The results are shown in parentheses. The linear regression lines calculated for each sample are shown in FIGS.

[0103]

[Table 6] Sample ID Cμg / g Sample ID Cμg / g 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

Example 20 Synthesis of a labeled antigen 4.6 mg (10.5 μmol) of RH2651
Was placed in a 5 ml pear-shaped flask and 100 m
Dissolved in 1 DMF. 3. In another test tube.
6 mg NHS and 8.8 μl DCC were
Dissolved in 100 μl DMF. NHS and DCC were added to RH2651 and stirred at room temperature for about 1 hour. Stirring was continued at 4 ° C. overnight. The next day, 10 mg of HRP
Dissolved in 7 ml of 0.01 M PBS, pH 7.2. The RH2651 / NHS / DCC reaction mixture was added to a Beck
Centrifuged for 3 minutes in man Microfuge E (Beckman Instruments). The supernatant was added dropwise to the HRP with stirring. The mixture was stirred at room temperature for 4 hours and then centrifuged for 3 minutes in a microcentrifuge. The supernatant containing the bound protein was applied to a Swif desalting column (Pier
ce Chemical Co. ). The protein was eluted with PBS and 3 ml fractions were collected. 280 nm
Fractions containing protein conjugates as determined by absorbance at were pooled and protein concentration was determined by BCA protein assay (Pierce Chemical Co.).

The binding of RH2651 to HRP was confirmed as follows. 10 microplate wells
coated with 100 μl of μg / ml 2651-HRP,
Stored at 4 ° C. overnight. After emptying the plates, the wells were blocked with 1% BSA in PBS. After blocking, RH2651 was analyzed by adding 1: 2 serial dilutions of protein-A purified 2651 IgG prepared according to Example 6 (starting at 1: 500) to the wells. After 1 hour incubation at room temperature, the plate is
Washed with Tween 20 PBS. Goat anti-rabbit IgG labeled with alkaline phosphatase was added and incubated for an additional hour at room temperature. Wash plate, p
-Nitrophenyl phosphate substrate was added. After a 15 minute run time, the absorbance was measured at 410 nm using a Dynatech MR5000 microplate reader.

Evaluation of the binding of RH2651 to horseradish peroxidase revealed that 1 μg of 265
A ^titer of 1: 32000 of ²⁶⁵¹ IgG against 1-HRP was shown. This indicated a sufficient binding reaction.

Example 21 Optimization of Coated Antibodies Microplates were coated with ²⁶⁵¹ IgG. Starting from column 1, 100 μl of 10 μg / ml ²⁶⁵¹ IgG
Was added to each well and a 1: 2 serial dilution was made across the plate. The plate was sealed and stored at 4 ° C until ready for use. After emptying, the plate was washed with 0.01% Tween 20 in PBS and 1% B in PBS.
Blocked for about 1 hour at room temperature with SA. After blocking, empty the plate and add 150 μl of PBS.
Was added to each well. Next, 50 μl of 265
1-HRP diluent was added to the plate starting at 10 μg / ml and the 1: 2 dilution was continued and added to the plate. After 1 hour incubation at room temperature, the plates were washed as described above and 150 μl of 1-Ste
p Slow-TMB (peroxidase substrate from Pierce Chemical Co.) was added to each well. 1
After an elapse of time, the reaction was stopped by adding 150 μl of 1N—H ₂ SO _4, and the absorbance of each well was measured using
4 with the tech MR5000 microplate reader
Measured at 50 nm. Control samples containing wells without coated antibody and wells without 2651-HRP were added. A ²⁶⁵¹ IgG coated antibody concentration of 0.625 μg / ml and a 2651-HRP concentration of 1.25 μg / ml were determined to be optimal concentrations for use with the 1-Step Slow-TMB substrate.

Example 22 Direct Assay Microplate wells were coated with 100 μl each of 0.625 μg / ml ²⁶⁵¹ IgG. One row was left uncoated as a control sample. Plate 4
C. overnight. The following day, plates are placed in PBS at 0.0
Washed with 1% Tween 20 and blocked with 1% BSA in PBS for at least 30 minutes. RH5992,
Standard solutions of RH2651, RH2703 and RH2485 were prepared from a 1 mg / ml stock solution. RH03
45 standard solutions at 0.1 ng / ml, 0.5 ng / m
1, 1 ng / ml, 5 ng / ml, 10 ng / ml, 5
Prepared at 0 ng / ml and 100 ng / ml. After emptying the blocking solution, 150 μl of the standard was added to the appropriate wells. 150 μl of PBS was added for the zero control sample. After 45 minutes incubation at room temperature, 50 μl of 1.25 μg / ml 2651-
HRP was added to each well and the plate was shaken to mix the contents. One column containing the antibody and standard did not add 2651-HRP, but instead
One liter of PBS was added to serve as a negative control sample. After a 45 minute incubation at room temperature, the plates were washed as described above, 150 μl of 2651-HRP was added to each well, and the plates were shaken to mix the contents.
One column containing the antibody and the standard sample has 2651-
Without HRP, 50 μl of PBS was added instead to serve as a negative control sample. After a 45 minute incubation at room temperature, the plates were washed as described above and 150 μl
Step-1 Slow-TMB was added to each well. After 2 hours at room temperature, 150 μl of 1N—H ₂ S
O ₄ was added to each well and Dynatech M
The absorbance at 450 nm was read on an R5000 microplate reader.

FIG. 16a shows a typical curve generated from the assay. The linear portion of the curve was adjusted to 0.01 ng /
Visualized between ml and 10 ng / ml. A typical standard curve in the linear range of the assay is shown in FIG. 16b using the reciprocal of the absorbance, with positive slope values. The detection limit was calculated to be 0.096 ng / ml, and the sensitivity was RH599
It was calculated to be 0.005 ng for 2. RH5992
Table 7 shows data relating to the compound and its analogs.

[0110]

TABLE 7 Test substance IC ₅₀ sensitive detection limits crossreactivity% ng / ml ng ng / ml RH2651 RH5992 RH5992 3.64 0.005 0.106 32 100 RH2703 2.10 0.002 0.031 56 173 RH2651 1.17 0.001 0.013 100 311 RH0345 16.94 0.255 5.10 6.9 21.5 RH2485 4.64 0.024 0.482 25 78.4

[Brief description of the drawings]

FIG. 1 is a view showing the elution characteristics of a protein from a DEAE ion exchange column.

FIG. 2 is a diagram showing a suppression curve for RH2703.

FIG. 3 is a diagram showing a suppression curve relating to RH2651.

FIG. 4 is a diagram showing a suppression curve relating to RH5992.

FIG. 5 is a diagram showing a suppression curve for RH0345.

FIG. 6 is a diagram showing a suppression curve relating to RH2485.

FIG. 7 shows external standard curves obtained for OBST and matrix blank samples.

FIG. 8 is a diagram showing a calculated linear regression line for a broccoli sample 20139-01.

FIG. 9 is a diagram showing a calculated linear regression line for a broccoli sample 20139-02.

FIG. 10 is a diagram showing a calculated linear regression line for a broccoli sample 20139-04.

FIG. 11 is a diagram showing a calculated linear regression line for a broccoli sample 20139-05.

FIG. 12 is a diagram showing a calculated linear regression line for a soil sample 151.

FIG. 13 is a diagram showing a calculated linear regression line for a soil sample 157.

FIG. 14 is a diagram showing a calculated linear regression line for a soil sample 175.

FIG. 15 is a diagram showing a calculated linear regression line for a soil sample 187.

FIGS. 16a and b show a direct assay curve and a direct assay standard curve.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Ellen Sharq Casal, Bluestone Mills Circle, Ashburn, Virginia, USA, 20147 43988 (72) Inventor Stanley Stephen Stabinski, 18969, Tell, Pennsylvania, USA Ford, Rising Sun Road 729 (72) Inventor Shagang Wu, Mallard Drive East, North Wales, 19454, Pennsylvania, USA 252

Claims (17)

[Claims] 1. The following formula bonded to a carrier material: Wherein R, R ¹ , R ² , R ³ and R ⁴ are each independently hydrogen, (C ₁ -C ₆ ) alkyl and substituted (C ₁ -C ₆ )
Alkyl, (C ₂ -C ₆ ) alkenyl and substituted (C ₂ -C ₆ )
C ₆ ) alkenyl, (C ₁ -C ₆ ) alkoxy, (C ₁ -C
₆ ) An immunogen comprising a compound of the formulas which are alkyloxy and halide. 2. The method according to claim 1, wherein the carrier material is a protein, a polysaccharide,
The immunogen of claim 1, wherein the immunogen is selected from the group consisting of a synthetic polymer or copolymer. 3. The immunogen according to claim 1, wherein R is —COOH, R ¹ and R ² are methyl, and R ³ and R ⁴ are hydrogen. 4. R is --CH ₃ COOH, R ¹ and R
² is methyl, immunogen of claim 1 R ³ and R ⁴ are hydrogen. 5. An antibody produced against the immunogen of claim 1. 6. An antibody produced against the immunogen of claim 3. 7. An antibody produced against the immunogen of claim 4. 8. The following steps: (A) providing a sample containing an unknown amount of diacylhydrazine; (B) providing the sample with a binding affinity for a diacylhydrazine antigen in the presence of an immobilized diacylhydrazine antigen. Contacting with the antibody to form a bound and unbound antibody-antigen complex; (C) unbound antibody-antigen complex is bound antibody-
(D) labeling the bound antibody complex with a detectable label; (E) causing a measurable change in the label; (F) measuring diacylhydrazine in the sample; , Diacylhydrazine or a derivative thereof. 9. The method of claim 8, wherein the detectable label is selected from enzymes, colored dyes, fluorescent materials, chemiluminescent materials, bioluminescent materials, and radioisotopes. 10. The method according to claim 8, wherein the detectable label is an enzyme-antibody complex capable of binding to the antibody. 11. The following steps: (A) providing a sample comprising an unknown amount of diacylhydrazine; (B) contacting the sample with an immobilized antibody having a binding affinity for a diacylhydrazine antigen, (C) contacting the diacylhydrazine antigen labeled with a detectable label with the uncomplexed immobilized antibody to form a labeled antibody-antigen complex; (D) in the label (E) measuring diacylhydrazine in a sample; a method for measuring diacylhydrazine or a derivative thereof, which comprises the step of: 12. The detectable label is an enzyme, a colored dye,
12. The method according to claim 11, wherein the method is selected from fluorescent materials, chemiluminescent materials, bioluminescent materials and radioisotopes. 13. The method according to claim 11, wherein the label is an enzyme-antigen complex capable of binding to the antibody. 14. The following components: (A) an antibody having a binding affinity for diacylhydrazine; (B) a label capable of binding to the antibody; and (C) producing a measurable change in the presence of the label. A kit for measuring diacylhydrazine, comprising: 15. The kit according to claim 14, further comprising a diacylhydrazine antigen. 16. The kit according to claim 14, wherein the label is an enzyme bound to an antibody having a binding affinity for the antibody. 17. The kit according to claim 14, wherein the label is an enzyme bound to a diacylhydrazine antigen having a binding affinity for an antibody.