JPH09154596A - Antibody specific to para-nitrophenoxy organic phosphorous compound, its production, and measurement of para-nitrophenoxy organic phosphorous compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new antibody specific to para-nitrophenoxy organic phosphorous compounds and enabling to simultaneously measure plural kinds of left agrochemicals by using the combination of a specific para-nitrophenoxyalkyl derivative with a biological polymer carrier as an immunogen. SOLUTION: The combination of a papa-nitrophenoxyalkyl derivative of formula I (R<1> is a functional group capable of binding to a carrier; X is CH2 , CH2 O, CH2 CH2 O, COO, CONH; n is an integer of 1-7; PhNO2 is para-nitrophenyl) with the carrier is used as an immunogen to obtain the new antibody which specifically reacts with two or more kinds of compounds of formula II [A is a group of formula III (R<2> is a 1-4C alkoxy, phenoxy; R<3> is a 1-4C alkoxy), a 1-4C alkyl)]. The antibody enables to measure several kinds of left agrochemicals in a measurement operation. The antibody is obtained by administering the combination of the compound of formula I [e.g. 1-(paral- nitrophenoxy)butyric acid] with a biological polymer carrier (e.g. ovalbumin as an immunogen into an animal such as mouse.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel antibody or an antibody fragment thereof that specifically reacts with a para-nitrophenoxy-based organophosphorus compound, particularly an organophosphorus-based pesticide having a para-nitrophenoxy structure. The present invention also relates to a method for producing this antibody, and further to an immunological measurement method for a para-nitrophenoxy type organophosphorus compound using this antibody and / or antibody fragment.

[0002]

2. Description of the Related Art Organophosphorus pesticides are typical pesticides used all over the world. In recent years, pesticides remaining in the environment and food have been attracting attention as a major social problem in terms of the effects on human bodies. Therefore, in order to ensure the safety of the environment and foods, it is first necessary to accurately measure the amount of pesticides remaining in them. Generally, the analytical method for residual pesticides is defined for each crop at the time of registration of the pesticide, and the pesticide is extracted mainly from the sample, and after purification, the content of the extract in the extract is analyzed using gas chromatography or liquid chromatography. The content is measured. Although this conventional measuring method has no problem in terms of accuracy, it has drawbacks in that the preparation of the sample is complicated and takes a long time, and the measuring device and equipment require a high cost. Since the number of analysis of pesticide residues is large, simplicity and speed are important in addition to accuracy. Further, it is required that the analysis can be easily performed without using an expensive measuring device. Conventionally,
As described above, the method of measuring the organophosphorus pesticide has been adopted by using gas chromatography, but the analysis requires a considerable amount of time and labor. In addition, the measuring device and equipment also require high cost.

On the other hand, in recent years, immunological detection methods have been applied to the measurement of components other than biological samples,
Various applications have been studied, but recently it has been applied to the measurement of some organophosphorus pesticides. For example, having a para-nitrophenoxy structure, the formula (I
V): an antibody against parathion represented by (C ₂ H ₅ O) ₂ P (= S) -O-Ph-NO ₂ (IV) (in the formula, -Ph-NO ₂ is a paranitrophenyl group). An immunological detection method using the method of Charles D. et al. Ercegovi
ch, Remo P. Vallejo, Russell
R. Gettig, Lyn Woods, Edward
rd R.D. Bogus, and Ralph O. Mum
ma, J.M. Agric. Food Chem. , 29,
559-563 (1981).

Similarly, it has a para-nitrophenoxy structure and has the formula (VII): (C ₂ H ₅ O) ₂ P (= O) -O-Ph-NO ₂ (VII) (where -Ph -NO ₂ -has the same meaning as above), and an antibody against paraoxon represented by
news W. , Hunter Jr. , AndDav
id E. Lenz, Life science, 3
0,355-361 (1982), M.A. Lover,
S. Krantz, and I.D. Herrmann, A
cta biol. med. germ. , 41,487
-496 (1982), Eliahu Heldma
n, Ayala Balan, OraHorowit
z, Sarit Ben-Zion, and Mich
ael Torten, FEBS LETTERS, 18
0,243-248 (1985), and Alan A. et al.
Brimfield, David E. Lenz, Ca
roline Graham, and Kenneth
W. Hunter, Jr. , J. et al. Agric. Foo
d Chem. , 33, 1237-1242 (198
5).

Further, regarding an inspection method for measuring an organophosphorus pesticide having a structure different from the para-nitrophenoxy structure but having a structure similar to that, for example, fenitrothion and related organophosphate esters, Japanese Patent Laid-Open No. 4-503760. in the formula _{(V): (CH 3 O} ) 2 P (= S) -O-B (V) ( wherein, B is a is 3-methyl-4-nitrophenyl group) and fenitrothion represented by Monoclonal and polyclonal antibodies for measuring similar organophosphates have been disclosed. However, all the known antibodies described in each of the above-mentioned documents are characterized in that the structure of the hapten used in the production of the antibody is an ester of an aromatic group and a phosphate group, and each of the above-mentioned antibodies is a hapten. It has been shown that they specifically react with the organophosphorus pesticides that were the basis of the design, but do not react with other organophosphorus pesticides or have extremely low reactivity. Therefore, it is difficult for the test method using these antibodies to easily and accurately measure many kinds of organophosphorus pesticides.

Further, Japanese Patent Publication No. 7-506814 discloses a new method for synthesizing a hapten for immunologically detecting an organic phosphate. However, this method is also characterized by containing an ester of an aromatic group and a phosphoric acid group in the structure of the hapten, as in the above-mentioned methods, so that the organophosphorus pesticide that was the basis of the hapten design It has only provided a means for the single and specific detection of organophosphates.

On the other hand, a plurality of organophosphorus pesticides having a paranitrophenoxy structure or a similar structure including methylparathion, ethyl-p-nitrophenylphenylthiophosphate (EPN), parathion and further fenitrothion are simultaneously and uniquely specified. Studies to detect positively have not previously been attempted. Simultaneous detection of multiple pesticides can be performed only once, and if at least one pesticide of the target pesticide group is contained in the sample, it can be detected. Therefore, it can be used as the first screening method when measuring multiple samples. Especially useful.

[0008]

Therefore, the present inventors have diligently studied based on an idea completely different from the conventional design method for hapten in order to simultaneously and specifically measure plural kinds of organophosphorus pesticides. As a result of research, instead of designing a hapten based on the organic phosphate ester structure of an organophosphorus pesticide that has been conventionally applied, by using a hapten having a completely different chemical structure, multiple types of paranitro We have succeeded in producing monoclonal and polyclonal antibodies that react specifically and simultaneously with a phenoxy-based organophosphorus compound. Furthermore, by utilizing these antibodies, multiple types of para-nitrophenoxy-based organophosphorus pesticides can be produced. It has become possible to make accurate measurements with only one immunological measurement.

Further, in the case of immunologically measuring a p-nitrophenoxy-based organophosphorus pesticide remaining in crops, soil, etc., an organic solvent is usually used to extract the pesticide from the test sample. . Therefore, it is desirable that the antigen-antibody reaction can be performed even in the presence of an organic solvent. Therefore, in the development of a rapid and simple operation process, the present inventors found an antibody showing resistance to an organic solvent from the above-mentioned monoclonal antibodies and polyclonal antibodies,
It was found that the use of this antibody enables immunologically rapid determination of a plurality of paranitrophenoxy-based organophosphorus pesticides even in the presence of an organic solvent, with only one measurement. The present invention is based on these findings.

[0010]

Accordingly, the present invention provides a compound of the general formula (VI): R ¹ -X _n -O-Ph-NO ₂ (VI) where R ¹ can be bound to a carrier. is a functional group, X is _{-CH 2 -, - CH 2 O} -, - CH 2 CH 2 O
-, -COO-, or -CONH-, and n is 1 to 7
Integer, in particular is 3, -Ph-NO ₂ is characterized by using a conjugate of a para-nitrophenoxy alkyl derivatives and carrier represented by a para-nitrophenyl group) as an immunogen, antibodies It relates to a manufacturing method. The present invention is also prepared by using a conjugate of a para-nitrophenoxyalkyl derivative represented by the general formula (VI) and a carrier as an immunogen, and the compound of the general formula (I): A-O-Ph-NO ₂ (I) wherein, a represents the general ^{formula: R 2 R 3 P (=} S) - ( wherein, R ² is an alkoxy group or a phenyl group having 1 to 4 carbon atoms, R ³ is C 1 -C Is an alkoxy group of 4 to 4) or is an alkyl group having 1 to 4 carbon atoms, and -Ph-NO ₂ has the same meaning as described above.]
The present invention relates to an antibody that specifically reacts with two or more compounds represented by. The present invention also relates to a fragment of the above antibody, which antibody fragment contains an antigen-binding site for the compound represented by the general formula (I). Furthermore, the present invention also relates to a method for immunologically measuring the compound represented by the general formula (I), which comprises using the above antibody and / or the above antibody fragment.

In the present specification, the term "paranitrophenoxy structure" means that a nitro group is substituted at the 4-position of the phenoxy group, and the benzene ring is further substituted with a substituent other than the 4-nitro group. The formed para-nitrophenoxy structure, such as a 3-methyl-4-nitrophenoxy group or a 2-chloro-4-nitrophenoxy group, is not included. Hereinafter, the compound represented by the general formula (I) may be referred to as a para-nitrophenoxy-based organic phosphorus compound.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for producing an antibody according to the present invention, that is, a method for producing a polyclonal antibody, a method for isolating a hybridoma producing a monoclonal antibody, a method for producing a monoclonal antibody, and an immunological measuring method will be described in this order. According to the present invention, by using the paranitrophenoxyalkyl derivative represented by the general formula (VI) as a hapten, a polyclonal antibody or a hybridoma producing a monoclonal antibody can be prepared. The preparation of the polyclonal antibody of the present invention, the isolation of the hybridoma producing the monoclonal antibody, and the preparation of the monoclonal antibody are carried out by a conventional method, for example, the method described in the Second Biochemical Experiment Course or the Immunobiochemical Research Method (edited by the Japanese Biochemical Society). Can be done according to. As used herein, the term “hapten” refers to a substance that has the ability to bind to an antibody, but does not have immunogenicity by itself, but expresses immunogenicity by binding to a carrier (shreper). means.

In the general formula (VI), R ¹ is a functional group capable of binding to the carrier. Here, the "carrier" means a substance (shredper) capable of expressing immunogenicity by forming a complex (conjugate) by binding with a hapten. Also in the present invention, a known substance that has been generally used as a shreper from the past can be used as a carrier. Therefore, as the carrier, for example, a biopolymer carrier such as a protein having a molecular weight of about 10,000 or more, preferably about 40,000 to 1,000,000 [eg, mammalian (eg, bovine or human) serum albumin, immunoglobulin, ovalbumin] is used. , Polylysine, keyhole limpet hemocyanin, or polysaccharides (eg, dextran,
Amylose, amylopectin); or cells [eg mammalian (eg bovine or human) erythrocytes or BCG
Fungus].

The above-mentioned "functional group capable of binding to a carrier" is not particularly limited as long as it is a functional group capable of binding to each of the above-mentioned carriers, and may be appropriately selected according to the carrier to be used. it can. For example, a functional group capable of binding to an amino group of a protein is a carboxyl group, an amino group, a hydroxyl group or a thiol group; a functional group capable of binding to a thiol group of a protein is a thiol group or an amino group; a polysaccharide. Examples of the functional group capable of binding to the hydroxyl group include a thiol group or an aldehyde group.

The binding of the para-nitrophenoxyalkyl derivative represented by the general formula (VI) and the biopolymer carrier (shredper) can be carried out by a conventionally known method. For example, when bovine serum albumin is used as the biopolymer carrier, the amino group of albumin and para-nitro can be prepared by the mixed acid anhydride method, glutaraldehyde method, m-maleimidobenzoyl-N-hydrosuccinimide method, carbodiimide method or the like. A conjugate with a phenoxyalkyl derivative can be prepared.

The paranitrophenoxyalkyl derivative represented by the general formula (VI) is preferably the general formula (VI-1): R ¹¹ -X _n -O-Ph-NO ₂ (VI-1) (formula Wherein R ¹¹ is —COOH, —NH ₂ , —SH, or —
Is OH, X, p nitrophenoxy alkyl derivatives n and -Ph-NO ₂ is represented in the same sense in which) as defined above, more preferably the formula (VI-2): HOOC- ( CH 2) 3 -O 1- (paranitrophenoxy) butyric acid represented by -Ph-NO ₂ (VI-2) (wherein -Ph-NO ₂ has the same meaning as described above) can be used.

Using an immunogenic complex formed from a paranitrophenoxyalkyl derivative represented by the above general formula (VI) and a carrier (shredper), particularly a biopolymer carrier, as an immunogen, antiserum (polyclonal The production of an antibody) or a monoclonal antibody is preferable in that an antibody capable of reacting with all or most of the compound represented by the general formula (I) can be produced. Since various compounds exist in organophosphorus pesticides, it is important to select an immunogen capable of obtaining an antibody capable of simultaneously measuring as many kinds of measurement targets as possible.

Using these immunogenic complexes, mammals or birds (eg, mouse, rat, chicken, etc.) can be immunized by the in vivo immunization method. For example,
The immunogenic complex is emulsified and mixed with an equal amount of Freund's complete or incomplete adjuvant, and subcutaneously administered to rabbits or mice (primary immunization). Thereafter, the same operation is performed at intervals of 2 to 4 weeks, and immunization is performed several times. Blood of the animal thus immunized can be collected to prepare the polyclonal antibody of the present invention as an antiserum. In addition, Ehrlich's ascites tumor cells are inoculated into the abdominal cavity of immunized mice or rats, and as a result, the polyclonal antibody of the present invention can be prepared from the accumulated ascites. That is, by using a conjugate of the compound represented by the general formula (VI) and the carrier, particularly the biopolymer carrier (in particular, serum albumin or immunoglobulin) as an immunogen, Antibodies capable of reacting with all or most of the compounds represented by

On the other hand, when the monoclonal antibody of the present invention is prepared, the above-mentioned immunization procedure [particularly, the compound represented by the general formula (VI) and the carrier, particularly the biopolymer carrier (particularly, Serum albumin or immunoglobulin)
Mammals or birds that have performed the conjugate with as an immunogen, for example, from a mouse, a few days after the final immunization operation, the spleen is aseptically removed, and spleen cells are prepared by crushing with a stainless steel mesh or the like, Used in the cell fusion process. As a myeloma cell (myeloma cell) which is the other parent cell of the cell fusion, various known cell lines, for example, p3.NS-1 / 1.Ag4.1 [Eur. J. I
mmunol. , 5,511-517 (1975)],
SP2 / 0-Ag14 [Nature, 276, 269
-270 (1978)], P3-X63-Ag8.65.
3 [J. Immunol. , 123, 1548-155
0 (1979)], NS0 [Methods in E]
nzymology, 73, 3 (1981)] and the like can be used.

The cell fusion can be carried out by an ordinary method, for example, using a known fusion promoter (for example, polyethylene glycol), and the ratio of cells to be used can be determined according to a conventional method.
For example, myeloma cells can be added to mouse spleen cells at a ratio of about 1/5 to 1/10. As the cell fusion medium, for example, commercially available polyethylene glycol for cell fusion (molecular weight 1500: manufactured by Boehringer Mannheim) can be used. The fusion can be performed by thoroughly mixing the immune spleen cells and the myeloma cells in the above medium.

Subsequently, cells other than the hybridomas are removed using a selection medium (for example, HAT medium), and the presence or absence of antibody production in the hybridoma culture supernatant is determined by, for example, ELI using the paranitrophenoxy-based organophosphorus compound.
The target hybridoma can be separated by measurement by the SA method. The hybridoma producing the antibody of the present invention can usually be cloned using a cell cloning method, for example, a limiting dilution method. When selecting a hybridoma producing a monoclonal antibody having resistance to an organic solvent, the paranitrophenoxy-based organophosphorus compound is added to an organic aqueous solution containing an organic solvent at various concentrations, and the monoclonal antibody is added thereto. After the addition, by confirming that the antigen-antibody reaction normally proceeds, the hybridoma producing the organic solvent-resistant monoclonal antibody can be isolated. As the organic solvent, it is preferable to select the solvent of the para-nitrophenoxy-based organic phosphorus compound to be measured. The isolated hybridoma can be subcultured in a known medium and can be easily stored in liquid nitrogen or the like for a long period of time.

The monoclonal antibody according to the present invention also comprises
It can be easily prepared by culturing a hybridoma that produces the desired monoclonal antibody. As the medium for culturing the hybridoma, in the case of in vitro, for example, any medium suitable for culturing under the conditions of 5% carbon dioxide and 37 ° C. can be used, and preferably, the Iscove's modified Dulbecco's medium (hereinafter A medium containing 10% fetal bovine serum (hereinafter referred to as FBS) (hereinafter, referred to as Iscove's medium) (hereinafter referred to as Iscove / 10% FBS medium) can be used. In the case of in vivo, it is preferable to culture in the abdominal cavity of a suitable mammal such as mouse.

The hybridoma culture solution, the ascites of a suitable mammal in which the hybridoma is intraperitoneally administered, or the antiserum containing a polyclonal antibody can be used as an antibody solution as it is. It is also possible to separate and purify the desired antibody using these antibody solutions as starting materials. Antibodies can be separated and purified using conventional methods that can be used to isolate and purify proteins. Examples of such methods include salting out with ammonium sulfate, ion exchange chromatography, molecular sieve column chromatography using a molecular sieve gel, affinity chromatography using a protein A or protein G binding polymer, dialysis and the like.

The antibody of the present invention specifically reacts with at least two compounds represented by the general formula (I). Various compounds (for example, organophosphorus pesticides having a para-nitrophenoxy structure) are present in the compound represented by the general formula (I), and the antibody of the present invention is represented by the general formula (I). Can specifically react with a plurality of compounds.
Antibodies having various specificities can be prepared by selecting an appropriate immunogen and / or a compound to be used for screening according to the target measurement object.

The antibody of the present invention has the formula (II): (CH ₃ O) ₂ P (= S) -O-Ph-NO ₂ (II) (wherein -Ph-NO ₂ is a para-nitrophenyl group). A compound represented by the formula (methylparathion), the formula (II
I): (C ₂ H ₅ O) (Ph) P (= S) -O-Ph-NO ₂ (III) (In the formula, Ph is a phenyl group, and -Ph-NO ₂ has the same meaning as above. A compound represented by the formula (EPN), and formula (IV): (C ₂ H ₅ O) ₂ P (= S) -O-Ph-NO ₂ (IV) (wherein -Ph-NO ₂ is It is preferably a monoclonal antibody that specifically reacts with a compound (parathion) having the same meaning as described above.

The antibody of the present invention specifically reacts with the compound represented by the formula (II) and the compound represented by the formula (IV), and further has the formula (V): (CH ₃ O ) ₂ P (= S) -OB (V) (wherein B is a 3-methyl-4-nitrophenyl group) is a polyclonal antibody that specifically reacts with a compound (fenitrothion). It is preferable. The compound represented by the general formula (I) includes, for example,
Some of the compounds represented by the formula (III) have optical isomers, and all the optical isomers are included in the compound represented by the general formula (I). When the antibody of the present invention is an antibody that specifically reacts with a compound having an optical isomer, it is preferably an antibody that specifically reacts with the racemic compound.

The antibody of the present invention includes an antibody fragment containing an antigen-binding site for the compound represented by the general formula (I), such as Fab, Fab ′, F (ab ′) ₂ or Fv. . These fragments can be obtained by, for example, digesting the antibody of the present invention with a proteolytic enzyme according to a conventional method, and then following a conventional method for protein isolation and purification.

Since the method for immunologically measuring the compound represented by the general formula (I) according to the present invention is carried out by using the polyclonal antibody and / or the monoclonal antibody or the antibody fragment, the paranitrophenoxy compound is used. It is possible to accurately measure an organic phosphorus compound of a system, in particular, an organic phosphorus pesticide having a para-nitrophenoxy structure. In the measuring method of the present invention, when an organic solvent-resistant monoclonal antibody is used, an accurate antigen-antibody in an organic aqueous system containing an organic solvent capable of sufficiently dissolving the paranitrophenoxy-based organophosphorus compound to be measured The reaction can proceed.

The measurement method according to the present invention uses a paranitrophenoxy-type organophosphorus compound, particularly a polyclonal antibody and / or a monoclonal antibody which reacts with an organophosphorus pesticide having a paranitrophenoxy structure, and preferably an organic solvent resistance Using antibodies (hence,
Except for the fact that the antigen-antibody reaction is carried out in the presence of an organic solvent), it can be applied as it is to a conventionally known immunological measurement method as it is. That is, it can be applied to enzyme immunoassay, latex agglutination, radioimmunoassay and the like. In the enzyme immunoassay, an antibody is enzyme-labeled, and among the enzyme-labeling methods in which the immobilized antigen and the paranitrophenoxy-type organophosphorus compound in the sample are competitively reacted, a method usually called a direct competitive method, or After the competitive reaction of the labeled antibody with the immobilized antigen and the paranitrophenoxy-type organophosphorus compound in the sample, the antibody bound to the immobilized antigen is detected using an enzyme-labeled secondary antibody. There is a method usually called a competitive method, or a method usually called an antigen labeling method in which an antibody is immobilized on a solid phase and the labeled antigen and the paranitrophenoxy-based organophosphorus compound in the sample compete with each other. Here, among the antibody labeling methods, the method of the present invention will be described below along with a measurement method usually called an indirect competitive method.

When a paranitrophenoxy-based organophosphorus compound, particularly an organophosphorus pesticide having a paranitrophenoxy structure, is to be measured by the measuring method according to the present invention,
For example, (1) a conjugate of a hapten [for example, a paranitrophenoxyalkyl derivative represented by the general formula (VI)] and a carrier, particularly a biopolymer carrier (eg, serum albumin or immunoglobulin) (2) A test sample (for example, soil, water, or food) is extracted with an organic solvent (particularly, a water-miscible organic solvent) when immobilized (hereinafter, referred to as immobilized antigen); A step of preparing a sample by diluting it with a buffer solution according to: (3) the solid-phased antigen, the sample, and a certain amount of the antibody of the present invention, that is, an antibody that reacts with a para-nitrophenoxy-based organophosphorus compound, In particular, a step of mixing with an antibody having resistance to an organic solvent (hereinafter referred to as the first antibody) to cause a competitive reaction; (4) The first antibody bound to the immobilized antigen and the paranitrophenoxy system in the sample Combined with organic phosphorus compounds (5) contacting the labeled anti-mouse antibody (hereinafter referred to as the second antibody) with the first antibody bound to the solid-phased antigen; (6) solid phase A second antibody further bound to the first antibody bound to the activated antigen and a second antibody not bound to the first antibody; and (7) one of the two separated in step (6), Preferably, it comprises the step of measuring the signal from the label of the second antibody bound to the first antibody.

The organic solvent which can be used in the method of the present invention is a solvent which can dissolve the para-nitrophenoxy type organic phosphorus compound. The water-miscible organic solvent is preferable because the antigen-antibody reaction can be performed after appropriately diluting the organic solvent extract with water. Examples of the organic solvent include alcohol compounds (for example, lower alcohols having 1 to 2 carbon atoms, particularly methyl alcohol and ethyl alcohol),
Mention may be made of ketone compounds (in particular acetone), or acetonitrile, or mixtures thereof.

In carrying out the method of the present invention, the labeled second antibody can be used to confirm or quantify the para-nitrophenoxy type organophosphorus compound. For labeling antibodies,
Known labels such as radioisotopes (eg, ³² P,
³⁵ S, or ³ H), enzyme (eg, peroxidase or alkaline phosphatase), vitamin (eg, biotin), fluorescent substance (eg, FIT)
C), or a chemiluminescent substance (eg, acridinium) or the like can be used.

The labeled second antibody can be added to the reaction system after the step of competitive reaction between the first antibody, the paranitrophenoxy type organic phosphorus compound and the solid phase antigen is completed.
That is, after the first antibody is competitively reacted with the paranitrophenoxy-based organophosphorus compound and the solid-phased antigen, the first antibody that has not bound to the solid-phased antigen is separated and removed, and then the labeled second antibody is added. When added, the labeled second antibody binds to the first antibody bound to the immobilized antigen.

In the method of the present invention, the first antibody bound to the solid-phased antigen is separated after the competitive reaction between the first antibody, the paranitrophenoxy type organic phosphorus compound and the solid-phased antigen is completed. Separation can be performed, for example, by filtration, centrifugation, or washing with a buffer. Furthermore, after the reaction between the first antibody bound to the solid-phased antigen and the labeled second antibody is completed, the labeled second antibody not bound to the first antibody is separated, and subsequently, for example, The signal from the label of the labeled secondary antibody bound to the 1 antibody is measured. When measuring a signal, it is preferable to change the reaction system containing the labeled second antibody to conditions suitable for signal measurement. For example, when a fluorescent or chemiluminescent substance is used as the label, the signal is measured under the condition that quenching does not occur.

[0035]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention. Example 1: Hapten having a para-nitrophenoxy structure
Preparation of a conjugate of a protein and a protein 1- (paranitro represented by the above formula (VI-2) having a paranitrophenoxy structure, which is one of the typical partial structures of organophosphorus pesticides, and a carboxyl group. After dissolving 25 mg of phenoxybutyric acid (hereinafter referred to as M-PA) in 2 ml of anhydrous dioxane, 50 μl of N-methylmorpholine
Was added and stirred at 10 ° C. for 10 minutes. Then, 20 μl of isobutyl chlorocarbonate was added little by little, and 2
Stirred for 0 minutes. On the other hand, after dissolving 80 mg of ovalbumin in 1 ml of distilled water, 2.6 ml of dioxane was added at 10 ° C.
Was added dropwise, and the pH was adjusted to 9 with 1N-NaOH. To this ovalbumin solution, while maintaining pH 9 with 1N-NaOH, the previously prepared M-PA solution was gradually added dropwise,
The reaction was carried out at 4 ° C for 4 hours. After completion of the reaction, it was dialyzed against a Dulbecco's phosphate buffer solution (hereinafter referred to as PBS (-)) to obtain a conjugate of M-PA and ovalbumin. The same operation as the above step was performed except that bovine serum albumin was used instead of ovalbumin to obtain a conjugate of M-PA and bovine serum albumin. The conjugate of M-PA and ovalbumin and the conjugate of M-PA and bovine serum albumin were used as an immunogen and an antigen for analysis after lyophilization.

Example 2: M-PA with ovalbumin
Immunization of Conjugate to Mice 100 μg of the conjugate of M-PA and ovalbumin was used as an immunogen, dissolved in 50 μl of PBS (−), mixed with an equal amount of Freund's complete adjuvant, and then subcutaneously inoculated into mice. The mice were immunized with. One month and three months later, 1/4 of the initial immunization was boosted.

Example 3: Preparation of Polyclonal Antibody Solution The ascites of the mice immunized in Example 2 above was collected by the following method to obtain a polyclonal antibody solution. First, one week after the booster immunization, Ehrlich ascites tumor cells were inoculated at a dose of 1 × 10 ⁷ cells / mouse. One week after that, the collected ascites fluid was collected and subjected to 10 rpm at 10,000 rpm.
Centrifuged for minutes, and the supernatant was used as a polyclonal antibody solution. The polyclonal antibody SP2-3 thus obtained was used in the following examples.

Example 4 Preparation of Monoclonal Antibody For use in cell fusion, the spleen was extracted from the mouse immunized in Example 2 3 days after the final immunization. First, the excised spleen was washed three times with Iscove's modified Dulbecco's medium (hereinafter referred to as Iscove's medium), and the spleen cells in the tissue were taken out into the Iscove's medium. This cell suspension was filtered through a stainless mesh to remove large solids, and the resulting spleen cells and mouse myeloma cells P3-X63-Ag.
8.653 and 3 were washed with Iscove's medium three times, and mixed at a cell number ratio of 5: 1 (spleen cells: myeloma cells) and centrifuged (1,200 rpm, 5 minutes) to precipitate the cells. Obtained. To this, 1 ml of a 50% polyethylene glycol (molecular weight 1,500) solution that had been warmed to 37 ° C. was slowly added to fuse the cells. After reacting for 1 minute, 10 ml of Iscove's medium was slowly added, and 1 ml of fetal bovine serum (hereinafter referred to as FBS) was further added to stop the cell fusion. The obtained cells were mixed with HAT medium (Iscove / 10% FBS medium at 13.6 μg / ml).
Hypoxanthine, 176 ng / ml aminopterin, and 3.88 μg / ml thymidine) and suspended in a 96-well microtiter plate at 2 × 1.
The cells were dispensed at a concentration of 0 ⁵ cells / ml and cultured at 37 ° C in the presence of 5% carbon dioxide for 10 to 14 days. After culture
Using the conjugate of M-PA and bovine serum albumin prepared in Example 1, the presence or absence of antibody production in each culture was screened.

The activity of the antibody was measured by the ELISA method. First, the conjugate of M-PA and bovine serum albumin was dissolved in PBS (−) at a concentration of 0.5 μg / ml, 100 μl / well of this solution was added to a 96-well microtiter plate, and then at 4 ° C. The antigen was solid-phased by standing still overnight. Next, the solution was replaced with 250 μl / well of 85 mM borate buffer (pH 8.0) containing 1% BSA and 62 mM sodium chloride (hereinafter referred to as blocking buffer), and the blocking reaction was performed at room temperature for 1 hour. After washing three times with 85 mM borate buffer (pH 8.0) containing 62 mM sodium chloride (hereinafter referred to as a washing solution), 75 μl / well of hybridoma culture supernatant was added and reacted at room temperature for 1 hour. After completion of the reaction, the plate was washed 3 times with the washing solution and then diluted with the secondary antibody [0.3% BSA and 6
A peroxidase-conjugated anti-mouse IgG antibody (secondary antibody) diluted 1,000-fold with 85 mM borate buffer (pH 8.0) containing 2 mM sodium chloride was added to 100
μl / well was added and reacted for 1 hour. After washing three times with a blocking buffer solution, a substrate solution of peroxidase [0.1 M acetic acid-sodium acetate buffer solution (pH 5.
5), 100 μg / ml tetramethylbenzidine (TM
BZ) and 0.006% H ₂ O ₂ ]] 100 μl / well was added, and after 10 minutes, 2N sulfuric acid was added to stop the color reaction. Wells containing cells producing antibody were selected by measuring the absorbance at 450 nm.

As a result, among the wells containing the culture supernatant, which had a positive reaction to the conjugate of M-PA and bovine serum albumin, those in which the inhibition reaction by the organophosphorus pesticide, methylparathion, occurred were indirectly competed. Secondary screening was performed using the inhibition ELISA method. First, as in the case of the above-mentioned ELISA method, the conjugate of M-PA and bovine serum albumin was adjusted to 0.
It was dissolved in PBS (-) at a concentration of 5 μg / ml, 100 μl / well of this solution was added to a 96-well microtiter plate, and then the antigen was immobilized at 4 ° C. overnight. Next, the blocking buffer was replaced with 250 μl / well, and the blocking reaction was performed at room temperature for 1 hour. After washing three times with a washing solution, 1 μm was added to an 85 mM borate buffer solution (pH 8.0) containing 1% methanol and 62 mM sodium chloride.
50 μl / well of a methylparathion (Wako Pure Chemical Industries) solution dissolved at a concentration of g / ml was added, and an equal amount of culture supernatant was immediately added and mixed, and the mixture was reacted at room temperature for 1 hour. After washing three times with the washing solution, 100 µl / well of the above-mentioned secondary antibody diluted 1,000 times with the secondary antibody dilution solution was added and reacted for 1 hour. After washing again with the washing solution three times, 100 μl / well of substrate solution of peroxidase was added to develop color, and 1
After 0 minutes, 2N sulfuric acid was added to stop the color reaction, and then 4
Absorbance at 50 nm was measured.

As a result of the secondary screening, the hybridoma in the well producing the desired antibody was subjected to cell cloning by the limiting dilution method. As a result of cell cloning, 3 strains of hybridomas producing a monoclonal antibody that inhibits the reaction with the M-PA / bovine serum albumin conjugate by methylparathion (18
-2 strain, 61-6 strain, and 70-5 strain) were obtained. The monoclonal antibodies produced by these hybridomas are referred to as 18-2, 61-6, and 70-5, respectively. Table 1 shows the immunoglobulin subclasses of the obtained monoclonal antibodies. Iskoff / 10 for those hybridomas
The cells were cultured in a% FBS medium, and each culture supernatant was used as a monoclonal antibody solution in the following experiment.

[0042]

[Table 1]

Example 5: Indirect competitive ELISA
Noclonal antibody 70-5 or polyclonal antibody SP
Reactivity of 2-3 with methyl parathion Monoclonal antibody 70-5 obtained in Example 4 above,
And the polyclonal antibody SP2 obtained in Example 3 above
For -3, the measurement sensitivity to methyl parathion, which is one of organophosphorus pesticides having a para-nitrophenoxy structure, was examined by the indirect competitive ELISA method shown below. The conjugate of M-PA and bovine serum albumin was added to 0.5
Dissolve in PBS (-) so that the concentration becomes μg / ml,
100 μl / well of the obtained solution was added to a 96-well microtiter plate and then left standing at 4 ° C. overnight to immobilize it. Next, the blocking buffer was replaced with 250 μl / well, and blocking was performed at room temperature for 1 hour. After washing each well three times with a washing solution, 50 μl / well of a solution of methylparathion in which the concentration was changed stepwise in an 85 mM borate buffer solution (pH 8.0) containing 1% methanol and 62 mM sodium chloride was added, and immediately. Antibody solution (monoclonal antibody 70-5 solution or polyclonal antibody SP2) diluted to an appropriate concentration with 85 mM borate buffer solution (pH 8.0) containing 62 mM sodium chloride.
-3 solution) 50 μl / well was added and mixed, and 1 at room temperature
The reaction was carried out for a time (hereinafter referred to as a competitive reaction step). Wash 3 times with the washing solution again, then use the secondary antibody diluent to
100 μl / well of 0-fold diluted peroxidase-conjugated anti-mouse IgG antibody was added, and the mixture was reacted for 1 hour. After washing with the washing solution three times, the substrate solution of peroxidase was allowed to develop color according to the procedure described in Example 4 above, and 450 n
m was measured.

The reactivity of the monoclonal antibody 70-5 with methyl parathion is shown in FIG.
The reactivity between -3 and methyl parathion is shown in FIG. From the result of FIG. 1, the monoclonal antibody 70-5 was about 0.
It was revealed that methylparathion can be quantitatively measured in the range of 5 to about 12.5 μg / ml.
From the results of FIG. 2, it was revealed that the polyclonal antibody SP2-3 can quantitatively measure methylparathion in the range of about 0.2 to about 50 μg / ml.

Example 6: Monoclonal antibody 70-5 and
And polyclonal antibody SP2-3 methyl alcohol resistance
For monoclonal antibodies 70-5 and polyclonal antibodies SP2-3 used in sexual Example 5, Example 5
The resistance to methyl alcohol was examined using the indirect competitive ELISA method described in (1). Each primary antibody was diluted at the same dilution rate as in Example 5, and at the same time, methyl alcohol was added so that the final concentration in the reaction solution of the competitive reaction step was a predetermined concentration in the range of 0 to 50%. In the reaction with methyl parathion, the result of methyl alcohol resistance of the monoclonal antibody 70-5 is shown in FIG. 3, and the result of methyl alcohol resistance of the polyclonal antibody SP2-3 is shown in FIG. From the results of FIG. 3, the monoclonal antibody 70-5 was
It was revealed that methylparathione can be sufficiently quantitatively measured at a methyl alcohol concentration of up to 40%, although the measurement sensitivity gradually decreases. On the other hand, from the results of FIG. 4, it is clear that the polyclonal antibody SP2-3 is relatively stable and can be measured up to a methyl alcohol concentration of 10%, but the sensitivity is rapidly decreased at higher concentrations. It was

Example 7: Derivatives of methyl parathion and
Cross-Reactivity with Related Pesticides Using the conditions described in Example 5 above, a reaction was made with a derivative or related pesticide instead of methylparathione, and monoclonal antibody 70-5 and polyclonal antibody SP2-
The cross reactivity of 3 was investigated. In this example, methylparathion, EPN (Wako Pure Chemical Industries), fenthion (Wako Pure Chemical Industries), parathion (Wako Pure Chemical Industries), propaphos (Wako Pure Chemical Industries), sulprophos (Hoechst), fenitrothion (Wako Pure Chemical Industries). ), Phensulfothion (Wako Pure Chemical Industries), p-nitrobenzoic acid (Wako Pure Chemical Industries), p-nitroanisole (Wako Pure Chemical Industries), and o
-Nitroanisole (Wako Pure Chemical Industries) was used. I of methylparathion of each antibody or its related compounds
The results of C ₅₀ value (μg / ml) and cross-reactivity value (%) are shown in Table 2. The IC ₅₀ value is defined as the concentration of a test compound required to inhibit 50% of the test compound, assuming that the absorbance is 100% when the reaction is performed without adding the test compound to be tested. The IC ₅₀ value (μg / ml) was used. The cross-reactivity value (%) is the IC of methyl parathion.
_It was defined as the percentage of the IC ₅₀ value of each test compound when the ₅₀ value was 100%, and was calculated by the following formula. Cross-reactivity value (%) = (IC ₅₀ of IC ₅₀ / test compound of methyl parathion) × 100

[0047]

Table 2 Monoclonal antibody 70-5 Polyclonal antibody SP2-3 Compound name IC ₅₀ cross-reactivity (%) IC ₅₀ cross-reactivity (%) Methyl parathion 1.5 100 5.7 100 EPN (*) 3.3 45. 5> 50 <11.4 Fenthione> 50 <3.0> 50 <11.4 Parathion 12.2 12.3 21.8 26.1 Propaphos> 50 <3.0> 50 <11.4 Sulprophos> 50 <3.0> 50 <11.4 fenitrothion> 50 <3.0 9.8 58.2 phensulfothion> 50 <3.0> 50 <11.4 p-nitrobenzoic acid> 50 <3.0> 50 <11 .4 p-nitroanisole 9.3 16.1 2.9 197 o-nitroanisole> 50 <3.0> 50 <11.4 [*]: As EPN, racemic Mi body was used.

The structural formulas of the test compounds used in this example are as follows.

[0049]

[Table 3]

[0050]

[Table 4]

As is clear from Table 2, the monoclonal antibody 70-5 hardly reacts with other derivatives having a structure different from the para-nitrophenoxy structure or organophosphorus pesticides. , Another compound having a para-nitrophenoxy structure, EPN, parathion, and p-nitroanisole showed significantly high reactivity, and a plurality of the above-mentioned para-nitrophenoxy-based organophosphorus compounds were simultaneously measured in one measurement operation. It became clear that it can be measured immunologically. On the other hand, the polyclonal antibody SP2-3 reacts not only with methylparathion but also with another compound having a paranitrophenoxy structure, parathion and p-nitroanisole, and further with fenitrothion. It was revealed that immunoassay can be performed simultaneously by one measurement operation.

[0052]

EFFECTS OF THE INVENTION Conventionally, a method of measuring an organophosphorus pesticide has been adopted by using gas chromatography, but the analysis requires a considerable amount of time and labor. In the immunological measurement method according to the present invention, for example, by using the monoclonal antibody 70-5, methylparathion, EPN, and parathion can be simultaneously measured by one measurement operation, or the polyclonal antibody SP.
By using 2-3, methylparathion, parathion, or fenitrothion can be simultaneously measured by one measurement operation. Further, methylparathion can be measured even under conditions containing methyl alcohol, for example, not only test water collected from rivers, paddy fields, or lakes, but also organic solvents from test samples such as crops and soil. It is considered that the extracted liquid can be easily measured. Therefore, by using the measurement method according to the present invention, it becomes possible to greatly simplify the measurement of the compound represented by the general formula (I) and shorten the measurement time,
A large number of specimens can be measured quickly and inexpensively.

[Brief description of the drawings]

FIG. 1 is a graph showing the reactivity of monoclonal antibody 70-5 with methyl parathion.

FIG. 2 is a graph showing the reactivity of polyclonal antibody SP2-3 with methyl parathion.

FIG. 3 is a graph showing methyl alcohol resistance of monoclonal antibody 70-5 in a reaction with methyl parathion.

FIG. 4 is a graph showing methyl alcohol resistance of polyclonal antibody SP2-3 in a reaction with methyl parathion.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/53 G01N 33/531 A 33/531 33/563 33/563 33/577 B 33/577 9282-4B C12N 15/00 C // (C12P 21/08 C12R 1:91) (72) Inventor Shunichi Takewaki 1-11-4 Higashi-Kanda, Chiyoda-ku, Tokyo Inside the company Yatron (72) Inventor Kita Hiroshi 1-11-4 Higashikanda, Chiyoda-ku, Tokyo Inside the company Yatron (72) Inventor Hideo Okawa 14-3-403 Asahigaoka-cho, Ashiya-shi, Hyogo

Claims (4)

[Claims] 1. General formula (VI): R ¹ —X _n —O—Ph—NO ₂ (VI) (wherein R ¹ is a functional group capable of binding to a carrier, and X is —CH _{2 -, - CH 2 O -,} - CH 2 CH 2 O
-, -COO-, or -CONH-, and n is 1 to 7
Is a whole number, and -Ph-NO ₂ is a para-nitrophenyl group), and the use of a conjugate of a para-nitrophenoxyalkyl derivative represented by the biopolymer carrier as an immunogen. Method. 2. General formula (VI): R ¹ —X _n —O—Ph—NO ₂ (VI) (wherein R ¹ is a functional group capable of binding to a carrier, and X is —CH _{2 -, - CH 2 O -,} - CH 2 CH 2 O
-, -COO-, or -CONH-, and n is 1 to 7
An integer, -Ph-NO ₂ are prepared conjugates of para-nitrophenoxy alkyl derivatives and carrier represented by a para-nitrophenyl group) as an immunogen, the general formula (I): A- _{O-Ph-NO 2 (I} ) wherein, a represents the general ^{formula: R 2 R 3 P (=} S) - ( wherein, R ² is an alkoxy group or a phenyl group having 1 to 4 carbon atoms, R ³ is either a group represented by a is) an alkoxy group having from 1 to 4 carbon atoms, or an alkyl group having 1 to 4 carbon atoms, -Ph-NO ₂ is the same as defined above]
An antibody that specifically reacts with two or more compounds represented by. 3. A fragment of the antibody according to claim 2, which has the general formula (I): A—O—Ph—NO ₂ (I) [wherein —Ph—NO ₂ is a paranitrophenyl group]. And A is a general formula: R ² R ³ P (= S)-(wherein, R ² is an alkoxy group having 1 to 4 carbon atoms or a phenyl group, and R ³ is an alkoxy group having 1 to 4 carbon atoms. Which is a group represented by the formula (1) or a C1-4 alkyl group]. 4. Use of the antibody according to claim 2 and / or the antibody fragment according to claim 3 in the general formula (I): A-O-Ph-NO ₂ (I) [formula In the formula, -Ph-NO ₂ is a paranitrophenyl group, A is a general formula: R ² R ³ P (= S)-(wherein, R ² is an alkoxy group having 1 to 4 carbon atoms or a phenyl group. R ³ is an alkoxy group having 1 to 4 carbon atoms) or an alkyl group having 1 to 4 carbon atoms].