JPS6020149A - Immunochemical measurement method and its reagent - Google Patents

Abstract

PURPOSE:To enable immunological measurement of a trace amt. of hapten in a specimen in short time with high specificness and good reproducibility by inducing an agglutination reaction between the hapten and single kind of a monoclonal sensitized carrier deposited on single kind of monoclonal antibody sensitized carrier. CONSTITUTION:The reagent to be used for this immunochemical measurement consists of (A) a carboxyl group-contg. water-soluble monoolefin high polymer compd. bound chemically with hapten or the chemically modified matter thereof or a hapten-deposited carrier formed by binding chemically said compd. with a high polymer latex carrier having about 0.01-2mu grain size and (B) single kind of monoclonal antibody-deposited carrier for said hapten to be combined with the above-mentioned hapten-deposited carrier. A drop of a suitably diluted test specimen is placed on, for example, a clean slide plate and a drop of the above- mentioned monoclonal antibody-deposited carrier is dropped thereon then a drop of the hapten-deposited carrier is dropped thereon. The three are thereafter thoroughly mixed under oscillation for several minutes and the reaction for prohibiting the agglutination thereof is observed, by which a trace amt. of the hapten in the specimen is detected with good accuracy.

Description

[Detailed description of the invention]

The present invention relates to a new type of immunochemical measurement method for haptens and a new type of measurement reagent suitable for use in the method. In particular, the present invention relates to a method and a test method for immunochemically measuring minute amounts of hepatic acid in a specimen such as blood, other body fluids, and body fluid components, and a test method thereof. ('Reality, reliability, accuracy, sensitive EL, and high specificity, without the trouble of false reactions,
A new type of tracing method that can accurately detect IR-r:, haptens in a specimen in a short time with good reproducibility, stability, and easy operation, and its use in this method. A new type of measurement test suitable for; More specifically, the present invention provides -L4) A carboxyl group-containing water-soluble monoolefin polymer compound to which a hapten or a chemically modified product thereof is chemically bonded, or the bond has a molecular weight of about 0.01 to about 2 A hazoden carrier chemically bonded to a polymeric latex carrier with a particle size of microns (.1 carrier and CB). A) and (
13) A method for immunochemically measuring butene in a skin sample as described above, characterized in that the agglutination inhibition reaction of both reagent components is measured. The present invention also provides (A) a water-soluble monoolefin-based polymer compound containing a carboxyl, J, (; A hapten-carrying carrier chemically bonded to a polymer latex J'TI body having a particle size of .01 to about 2 microns, and (13) a single-weight monoclonal antibody Jl[] to the above-mentioned hapten from the J'TI carrier. It also relates to an immunochemical measurement reagent for butene, which is characterized by the following: Conventionally, it has been used to detect biologically active substances present in specimens such as blood, urine and other body fluids and body fluid components. T
The method of measuring by immunochemical means has been known for a long time. Such an immunochemical measurement method uses, for example, red blood cells as a carrier, sensitizes them with antigens or antibodies, and reacts them with the antibodies or antigens being subjected to immunochemical agglutination or agglutination inhibition. A method of causing a reaction and measuring the 4 substances on the machine is also already known. In addition, instead of hemocytes as carriers, non-biological particles such as synthetic resin latex, bentonite, J-lodion, cholesterol crystals, quartz, etc. can be used as solid jLJ in immunochemical reactions.
It is also known to be used as a body (for example, %Kokai No. 50-82230, Publication No. 4). The existence (qualitative) of an antigen in a specimen or its presence can be determined by immunochemical techniques that utilize such antigen-antibody reactions.
゛> The antibody-sensitized carrier that has been conventionally and commonly used for measuring and detecting K (stationary) is Ha? It was a reclonal antibody sensitized carrier. In 1975, Kfilstein et al. isolated mouse myeloma #ill l1iq and antibody-producing cells in the cells.
? lil I! i・IFl? It together, this, I'll+
Since the successful production of monoclonal antibodies from cells, the cell θ
11 (With the remarkable technological progress in the field of synthetic technology, it has become easier to form a single monoclonal antibody strain and use it to produce monoclonal antibodies. Proposals have been made regarding immunochemical measurement methods for antigens using or more different f = n F monoclonal antibodies can be used to target the same antigen [7]. An antigen hole 1:1 method using 3S has been proposed.
This proposal is completely different from the known phenomenon in which conventional antibodies, that is, polyclonal antibodies, react with the corresponding antigen and produce a precipitate. It has been described that carrier particles such as red blood cells, latex spheres, total chromatographic particles, etc. coated with null antibodies do not agglutinate in the presence of the corresponding antigen. In this proposal, unlike conventional knowledge, monoclonal antibodies tend to bind to the corresponding antigen and form precipitates. Therefore, this proposal requires the use of at least two types of blIjlt monoclonal antibodies as described above. It is specified in the antigen quantification method. Furthermore, this proposal also requires the use of such multiple types of heterologous monoclonal antibodies, (1・I [-i sensitivity and ! It is not always possible to get good results or even to be completely ineffective, so it is important to try every possible way of combining (r-1: what not to do). Write down j71i L
,ing. There are several kinds of such r'! As a similar proposal that requires the use of type A monoclonal antibodies,! 1-〒Kaisho 5
The proposal of No. 7-118159 is also known. In the conventional proposals for immunochemical measurement methods for antigens using these monoclonal antibodies, unlike the conventional polyclonal antibodies, monoclonal antibodies are compatible with monoclonal antibodies. Naturally, the use of different F, -r monoclonal antibodies with antibodies that do not bind to the antigen and produce a precipitate, but rather produce a precipitate, does not result in the formation of a precipitate. It is common that the use of several scales of monoclonal antibodies is essential.However, due to the force, as described in the former proposal, agglutination of the kinoclonal antibody and the corresponding antigen occurs. However, even if a carrier is sensitized with multiple types of heterologous monoclonal antibodies [7], good results may not always be achieved in terms of measurement sensitivity and specificity [7]. There were deficiencies and even serious technical shortcomings in that they did not even produce usable external agglomeration and were even completely ineffective. Naturally, the more a heterologous monoclonal antibody is used, the more the advantage of high specificity due to the use of a monoclonal antibody is lost, which is an unavoidable technical problem. In other words, monoclonal antibodies are antibodies that have the ability to specifically recognize one specific site in an antigen molecule.
The more monoclonal antibodies of two or more types are used, the more specific sites of N5 n6 can be targeted at the same time.
As a result, the use of monoclonal antibodies resulted in a high
The technical problem of reconciling this problem is that more and more of the advantages of heterosexuality will be lost. Furthermore, in the case of using conventional polyclonal antibodies that can target multiple sites on the antigen molecule (as mentioned above, monoclonal antibodies can be used to target multiple sites on the antigen molecule). Since it is not recognized, I'44jt 4i1 in the agglutination reaction compared to the case of using Hq reclonal antijuice.
,il,〒、;It is predicted that it will be very weak! , +J, and in fact, as mentioned above, the use of monoclonal antibodies
,,, In the tξΣ plan, when using a monoclonal antibody of type A, it binds to the antigen and forms a precipitate.
Inode Kushigo City! ? , teaches that the use of Jili monoclonal antibodies is essential, and furthermore, in the former proposal mentioned above, if agglutination still does not occur even if 7U'4 types of different 11 monoclonal antibodies are used. There is a technique '0:' Discloses that there is a certain flaw. The present inventors have attempted to achieve the above-mentioned compatibility when using monoclonal antibodies in immunochemical antigen measurement methods. Research has been conducted to develop methods that can solve difficult technical problems or technical deficiencies. As a result, in the case of a hapten-carrying carrier in which no-butene is supported on a carrier, a single type of monoclonal antibody against the no-butene is supported on the carrier, which is completely different from the above-mentioned conventional knowledge in the use of monoclonal antibodies. A total of 11 agglutination reactions occur between the single type of monoclonal antibody sensitized carrier, and thus it is possible to utilize multiple types of different types of monoclonal antibodies, which was essential to the conventional knowledge in the use of monoclonal antibodies. I gained new and unexpected knowledge that there is no need to do this at all. As a result of further research based on this completely new knowledge regarding the use of monoclonal antibodies, we have developed (A) a water-soluble monoolefin-based polymer compound containing a carboxy (carboxy) group, which is chemically bonded with a hapten or its chemically modified product; or (B) a butene-supporting carrier comprising C, in which the bond is chemically joined to a polymer latex extrusion having a particle size of about 001 to about 2 microns σ), and (B) J4'+ for the upper Be butene; A new test consisting of a combination of monoclonal antibodies JEJ and JU of species [7 different types, using 19:t! ! x In the sample) and butene (A)
) and (13) both tests J, a new type 0 immunity test that measures the agglutination inhibition reaction against the m1 reaction of 1≦components.
A chemical method for measuring butene in the above sulfur sample is proposed]
J1. possible, significantly better certainty, reliability, J'+1 degree, sensitivity and significantly higher antigen! 1.It has excellent P5 activity and stability without the trouble of false reactions due to its isomerism.It can immunochemically remove trace amounts of butenes in a sample in a short time with easy operation. Moreover, the above-mentioned remarkable effects have the advantage that the type of monoclonal antibody against the hapten of interest is virtually unaffected, and thus can be used in a wide variety of applications.・It was found that it can be applied to an immunochemical measurement method for butene. Therefore, the purpose of the present invention is to provide a new type of immunochemical measurement method for butene using a monoclonal antibody and its use in the method. Trying out a new type of measurement suitable for; 1
It is to provide the mi. The above objects and many other objects and advantages of the present invention will become clearer from the following description. (In the present invention) Butene (incomplete antigen) This means excluding antigens that have the ability to produce antibodies by themselves, that is, all photoantigens, and refers to low-molecular compounds that do not have antigenicity in themselves, but have anti-yy and anti-yy properties.
1. Binds to a polymeric compound having antigenic properties, such as those in Type 1.

[7] A substance that exhibits antigenicity and is reactive with antibodies produced by immunization with antigenic substances 1 and II. As such haptens, components present in living bodies (including physiologically active components and their substitutes, 111 products) and system substances administered to living organisms and their 1q1 products are the objects of the present invention as such haptens. It is the main point as a butene.
5 and JP-A No. 55-52946 discloses that a carboxyl group (i)-hydrohydric monoolefinic polymer compound having a carboxyl group chemically bonded with a (σ) hapten or a chemically modified product thereof has approximately (1, Ql to about 2 micron particle size 1, - turned into molecular latex!! 74 bonding)
1) Spicy as a package: i, na (te epidemic chemical measurement test, and the (α) no fu0ten J'Q !-' + latex, (/J) ... butene antibody E.; Immunochemical measurement method for butene (Japanese Unexamined Patent Publication No. 55-52945) or (C) chemically bonding hasithene or a chemically modified product thereof to a 17-medium carboxyl group-containing 7J< soluble monoolefin. Immunochemical assay results for hashiten obtained from the system polymer compound M:J and one hapten-conjugated compound (in which a hapten antibody is chemically bonded to a carrier or sensitized) Jj≦(Unexamined Japanese Patent Publication No. 55 -52946), furthermore, the above (a) and (b), or (C) and the above (a) with butene (using mosquito, in the specimen).
, (b) or (C), (characterized by measuring the δ''/recruitment inhibition reaction of both test strips), an immunochemical measurement method for butene has been proposed.In these 42 proposals, does not exclude monoclonal antibodies as antibodies in (b) and () above, but does not specifically address the use of such monoclonal antibodies, nor does it provide any specific examples. In the present invention, as for the above proposition, (b) and ( in j=, this proposal does not specifically disclose and also unlike the previous proposal of using monoclonal antibodies mentioned above, ( B) By selectively utilizing the monoclonal antibody-conjugated monoclonal antibody against the above-mentioned Hashiten, it is possible to obtain significantly higher certainty, reliability, sensitivity, and specificity of the antigen. , without the trouble of generating false reactions, is excellent in its properties, stability, and easy sweeping,
A trace amount of hapten in a 71·ν coating can be immunochemically measured in a short time. The water-soluble monoolefin-based polymer compound containing carboxyl-1, which is used in the present invention and in which a hapten or a chemically modified product thereof is chemically bonded, or the bond is about 0.01 to about 2 1. Hapten or its chemically modified product and modification method, chemically bonding the hapten or its chemically modified product to be used in "Hapten supported chemically bonded to a polymeric latex carrier with a micron particle size" Carboxyl group-containing water-soluble monoolefin polymer compound (for convenience, referred to as CWp), the CI?”p
A method for chemically bonding the hasithene or a chemically modified substance thereof to a compound and a compound thereof, further a means for chemically bonding the compound and a polymer latex carrier, and a hapten 4 thus formed.
11-supporting carriers are described in detail in the above-mentioned Japanese Patent Laid-Open No. 55-52945 filed by the same person, and can be used in this paper. Examples of haptens to be used include Examples include the following: (1) Steroidal hapteny (i) Follicular hormones such as evaestrone, estradiol, estriol, estetrol, equilin, equilenin, etc. (ii) For example, prog-9sterone; zolegnan Diols; pregnanetriols; 9-progestins into synthetic progestins such as 19-trueethisterone and 6-tachlormadinone; Male hormones, (1v) For example, adrenal cortical hormones such as cortisol, cortisone, deoxycorticosterone, aldosterone, tetrahydroaldosterone, and tetrahydrocortisol, (v) Vitamin J); cholesterol; e.g.
:I: Bile r such as cholic acid, desoxycholic acid, chenocholic acid;:? : Cardiotropic steroids; saponins; other steroids such as saponins and other steroids. (n) Physiologically active amines (i) Catecholamines and their metabolites such as epinephrine, norepinephrine, degumine, and ephedrine; (ii) Morphin, codine, heroin, morphin glucuronide, cocaine, mescaline, and Physiologically active alkaloids such as nogperine, narcotine, yohimbine, reserpine, ergotamine, strychnine; (iii) LSI), amphetamine, mesolonomete, methamphetamine, etc. (m) Other butenes? Low molecular weight peptides without antigenicity such as IL, Lll-R11; Thyroid fragments such as soiodothyronine, triiodothyronine, and thyroxine? hormone;
Prostaglandin E2, prostaglandin E3, prostaglandin p'lα~ha) prostaglandinso7
p;fi,;vitamin A1 vitamin Bg511 ()
For vitamins such as vitamin E1 (・vitamin 73.. B2, Bo, IJ, 2, etc.), vitamin E1; penicillin, actinomycin,
Antibiotics such as chloromycetin and tetracycline. Examples of haptens to be used and examples of haptens used include components that exist in living organisms, substances and their products, etc. Although it is possible to give examples of the same, '4V 'j'i'i l shout's ``Hageten it'', +i is the above example of ``Hanotentsu (1
1. ・It is not something that can be determined. In the present invention, hapten &'''' as exemplified above is chemically bonded to CFP either as it is or as a chemically modified chemical substance.As a method for chemically modifying such hapten, Various methods are conventionally known and can be used in the present invention. Modification methods may be employed. Modification methods for such halogens include chemical methods of introducing carboxyl groups, primary or secondary amino groups, or hydroxyl groups, especially carboxyl groups or primary amine groups into the hapten. Modification methods are preferred. Examples of such methods include the following modification methods: For haptens having a carbonyl group, a xyl group can be introduced by converting the carbonyl group into carboxylmethyl oxime (e.g. Journal of
Biological Chemistry, Vol.
. 234.1090-1094 (1959,
ids, L vol. 9, pp. 357-375 (1972)). It is well known that when the oxime of a carbonyl compound is converted to 57 atoms, it becomes a primary amino compound, which can also be used as hasitene. However, water [having a V group] and butene can be converted into carboxylic acid/methyl ether by reacting with monochloroacetic acid (e.g., 5science, Vol. 168, pp. 1347-1348 (1970)), or is reacted with succinic anhydride to form hemisuccinate (e.g., Jo
nbnal of C11nical Jln, doc
rinology. 33, pp. 775-782 (1971)) There are side dishes. For No-puan having a phenolic hydroxyl group, diazo coupling of, for example,
Introducing xyl groups (e.g. 5te-roids, l
8, pp. 555-563 (1971)). Furthermore, glucuronide, which is a metabolite of steroids, can utilize its carboxyl group. For example: Journal of 5teroids, Bio
-chemistry, vol. 3, pp. 275-288 (1
(972)), if the carboxyl group cannot be used directly, it can be converted into an amine compound by reacting with a suitable soamine derivative. Butene with a secondary amino group can be converted into a primary amine derivative by, for example, alkylating the amine group with an N-protected aminoalkyl compound and then removing the protecting group by IF. Possible (Example: FE13 S Lett
ers, vol. 36, pp. 339-342 (19, 73)),
Alternatively, by reacting with U bromoacetic acid ester and then hydrolyzing it, a carbyl group can also be produced (e.g., Chemicrtl a, n, dpjr, a).
, rmacev, ticat) Jtr, 1lali
n,, 2 5 volumes + 3 3 8 -840 pages (1
977)) is also possible. Haptens that do not have a suitable functional group can be easily synthesized by a biological means such as hydroxylation reaction by mimetic organisms, and then by a double method to obtain the desired functional group.
You can also. Further, as the carboxyl group-containing water-soluble monoolefin polymer compound (CJVp) used for chemically bonding hapten or its chemically modified product as exemplified above, water bathable Any monoolefin-based polymer compound may be used.Here, "
1, product+'ll means 1 part of the said disease-prone compound added to 2 parts of 1,000 ml distilled water,
L91] Na! It means to form f'~1. The purity + fT degree of the polymerized -8 product is J, ': pli: lower limit 1
As long as j~ is added, the common solution Py can be as large as it is. In addition, the average M weight molecular weight of the polymer compound is 3, about 10
The number may be 8 to 10 7 or more, and usually tens of thousands to several million can be suitably used. In addition, such a polymer compound has a hydroxyl group (-
01#), and these functional groups participate in chemical bonding with the functional groups of no-butene or its chemically modified product and the functional groups of the polymer latex carrier described below. , also adds water solubility to the polymer compound. The polymer compound used in this invention is considered to be a physiologically inert substance, and generally has no antigenicity.
It's something you don't do. Examples of such polymeric compounds include, for example, homo- or copolymers of acrylic acid or methacrylic acid; copolymers of maleic acid and vinyl acetate or saponified products thereof, maleic acid and, for example, vinyl alcohol, lower alkyl Copolymers with vinyl ether, acrylic acid or lower alkyl esters thereof, methacrylic acid or lower alkyl esters thereof, or optionally hydrolyzed products thereof may be mentioned. These polymeric compounds are copolymers of, for example, acrylic acid or methacrylic r,t with, for example, pre-acrylic β-hydroxyethyl ester or acrylamide, or contain the above-mentioned monomers as constituent units. It may also be a terpolymer. In the present invention, the chemical bond between no-butene or its chemically modified product as exemplified above and the polymer compound as exemplified above can be carried out through an amide bond or an ester bond. Preferably, an amide bond reaction is used. Such an amide bond reaction or ester bond reaction can be performed using a method known per se. For example, if hapten or its chemically modified product is represented by no-butene, if no-butene has an amino group and CU7p has a carboxyl group, or vice versa, halogen and CWp. can be chemically bonded to an amide bond by the method described below. Method of bonding hapten and cny p with amide bond (1) Carbodiimide method This is a method in which an amide bond is formed by dehydration condensation between an amine group and a carboxyl group. This objective can be achieved by adding an excess of the carbodiimide compound and reacting at room temperature or under water cooling. The carbosomide itself is converted to a urea derivative. -Ni12+-COOII + R-N =C=N-R
-+ , -NJI-CO-10R-NIICON11-R
The reaction solvent is not particularly limited as long as it does not itself have a functional group that participates in the reaction, and examples of usable solvents include ethyl acetate, tetrahydrofuran, dioxane, trimethylformamide, and chloroporm. The system may contain water in any proportion, and the reaction may be carried out in an aqueous solution. There are six types of carbodiimide compounds to be used. Dicyclohexylcarbosoimide is most often used for the reaction in I; and dicyclohexylcarbosoimide, which is water-insoluble, is used for the reaction in I-, for example, ←1-ethyl-3-(3-methylaminepropyl)carbodiimide hydrochloride, etc. Water-soluble carbodiimides such as can be used. (2) Carbonyl soimidazono+-v6calyf
The required carbonyldiimidazole may be added to a solution of both components by a method of causing dehydration between an amine group and a carboxyl group in exactly the same manner as the J imide method. Since this test J14 is sensitive to moisture and dissolves immediately with water, it is preferable to use solution 111 (which does not contain F1). (3) Mixed acid anhydride method The carboxyl group is replaced with chloroformate In the presence of a base, a so-called mixed acid anhydride is formed, which easily reacts with an amino group to form an amide bond. As a lower alkyl ester, isobutyl chloroformate is often used, and as a specific base, tertiary amines such as triethylamine, N-methylmorpholine, etc. can be used.As a reaction solvent, the same as mentioned in the carbodiimide method can be used. However, since mixed acid anhydrides are unstable in water, it is best to be careful not to use water-containing ones when forming mixed acid anhydrides.The reaction temperature is as follows: First, mix at a low temperature of, for example, -10° to -20° (;?
A preferred example is a temperature condition in which the reaction is continued in chamber 1 after stirring to form a water layer and then adding the amino component. When adding the amine component, water is added, <
11. (4) Active ester method When carboxyl group)1; is converted into an ester of a compound with large child attraction property, , the electron density on the carbonyl thread increases, and as a result, it attacks highly basic functional groups such as amino groups to form an amide bond.This is the principle of the active ester method, and as an active Nisdel. For example, phenols such as paranitrophenol, 2,4-dinitrophenol, pentachlorophenol, thiophenol, naphthol, and 8-noydroxyquinoline?+'
34 compound; For example, N-hydroxy compound such as N-hydroxysuccinimide, N-no-i)'-oxypiperiline; For example, an alkyl ester such as cyanomethyl can be used. As a solvent, a carbosimide (5) Amide method When a carboxyl compound is converted into an ester and reacted with hydrazine, it becomes an acid hydrazide.
The action turns into an acid amide, which reacts with an amino group to form an amide bond. Although the classical method of reacting hydrazide with sodium nitrate in dilute hydrochloric acid to form an acid azide, step by step, and then reacting with an amine component in a suitable organic solvent, is also available, Alkyl nitrite ester (for example, h+) in the presence of aqueous chloride (which may also contain water)
It is treated with H t-butyl nitrite, i-amyl nitrite, etc. to obtain azide, and a single t';i! An amide bond can be formed by reacting with an amine component without . (6) Acid chloride method It is also possible to employ a general method in which Cal converts a xyl compound into acid chloride and reacts it with an amino compound to generate an amide bond. To produce acid chloride, there are a direct method in which a carboxyl compound is reacted with phosphorus pentachloride, thionyl chloride, phosphorus oxychloride, etc., and an exchange reaction method in which it is reacted with, for example, oxalic acid chloride. The reaction with the amine compound is carried out by the so-called "Schotten-Bauman method" in which an alkali is added to water, or if necessary, in an inert solvent such as benzene in the presence of a 43i base such as pyridine or triethylamine. There are various ways of reacting, which can be used in the present invention. +7) DI) P A method There is also a method in which an amide bond is formed by adding sophenylphosphoryl azide (DPPA) and then an organic base such as triethylamine or N-methylmorpholine to a solution of a carboxyl compound and an amine compound. Can be adopted. In this case, for example, dimethylformamide is preferably used as the solvent, and the temperature may range from water cooling to room temperature. Although the purpose can be achieved using any of the chemical bonding methods described above (11 to (7) in which hapten and ctvp are bonded together through an amide bond), when applied to the present invention, bald may be unstable depending on the other substituents it has, so those that require too harsh conditions should be avoided.The most preferably used methods are 0) carbodiimide method, (7) D P P
This is method A. CIV p and appropriate measures to introduce 1. In a solution of hapten, there are two to two equimolar or i^'iy haptens.
When I>K excess carbosomide or 1tDPPA is added, a nitride base can be added and the reaction is carried out. After the reaction, the entire reaction solution is analyzed against water in a cellophane tube at 100°C. Unreacted haptens, states, byproducts, etc. will escape to the outer solution, so the inner solution should be
It is possible to obtain the desired halogen or its modified product-Clr'p conjugate by condensing or drying. Furthermore, the chemical bond between hasitene or its chemically modified product and the CTrI p polymer compound is such that the hapten or its chemically modified product has:
(If CIVp has a carboxyl group or vice versa, it can also be carried out by chemically bonding the hapten and CI7;'P with an ester bond. Bonding the hapten and c n″p with an ester bond Method: In the case of monoester 1,17 method, when the hapten or its chemically modified product has a water group and cr:'p has a carboxyl group, the carboxyl group is reacted with, for example, thionyl chloride. Convert to acid chloride, or if CI?-p is a copolymer containing maleic anhydride, for example, react it with balagetene to obtain a hapten-cn]"p bond with an ester bond. On the contrary, when haftene or its chemically modified product has a carboxyl group and CIVP has a hydroxyl group, depending on the nature of the hapten, it can be converted to a reactive B-friendly compound such as an acid chloride. In some cases, it may not have sufficient stability, and in this case, it is difficult to form an ester bond.The hapten-C117p conjugate thus obtained can be a conjugate consisting of many combinations of hapten and CWP. However, if we give a concrete representative example,
For example, the following combinations can be exemplified. 17-amino-], 3.5(10)-estratrien-3-ol-bonded polyacrylic acid, 17-amino-!
, 3.5(10)-estratrien-3-ol-bonded vinyl methyl ether maleic anhydride ri copolymer, estriol-16-glucuronide-bonded polyacrylic acid, estriol-16-glucuronide-bonded vinyl methyl ether maleic anhydride copolymer Polymer, estriol
16,17-sohemisuccinate-bonded polyacrylic acid, pregnanediol-3-glucuronide-bonded polyacrylic acid, pregnanediol-3-glucuronide-bonded vinyl methyl ether maleic anhydride copolymer, pregnanediol-3-glucuronide 4″11i polymer Acrylic acid, pregnane triol-3-glucuronide-bonded vinyl methyl ether maleic anhydride copolymer, 3α, 11β
, 17α, 21-tetrahydroxypregnane-20-
on-3-glucuronide-bonded polyacrylic acid, 3α, 11β+17α, 21-tetrahydroxypregnane-20-one-3-glucuronide-bonded vinyl methyl ether maleic anhydride copolymer, Cal d? Gysimethylmorphine is old 'cl'F ;re
IJ acrylic acid, carboxymethylmorphine-bonded vinyl methyl ether maleic anhydride copolymer, ethiocholanolone-3-hemisuccinate-bonded polyacrylic acid, thyroxine-bonded vinyl methyl ether maleic anhydride copolymer, zyroxine-bonded poly-reacrylic Examples include metanephilin-bonded polyacrylic acid, metanephilin-bonded vinyl methyl ether maleic anhydride compound, etc. (A) Hapten 4-functional carrier tj1 in the present invention
, the hapten or ti that can be obtained as described above
tChemically combine the chemically modified product/this CI! '
/l is further transformed into a reactive polymer latex carrier! 1′
It can be obtained by combining the polymer latex with an average particle size of about 0.01 to
A material having a diameter of about 2 microns and a functional group capable of reacting with the glare spacer is used. Those with an average diameter of about 0.05 to about 1.5 microns! To the temple! JJ′
1. It is 'fi'. The reactive polymer latex carrier may contain a carboxyl group, a primary amine group, or a carboxamide group (-CONII2) as a functional group, and the substrate may be, for example, polystyrene, styrene-butatsuene copolymer, styrene-divinylbenzene. Examples include polymeric latex carriers made of copolymers, polyvinyltoluene, vinyltoluene-tert-butylstyrene, and the like. Such latex carriers are commercially available under various trade names, and any of these polymeric latex carriers can be used. The base material of the polymer latex carrier is, of course, not limited to the polymers or copolymers exemplified above. Polymer latex carrier has primary Amitsuga as a functional group;
If the CH7p has a polymer latex carrier, it can be reacted with the carboxyl group of the CH7p to chemically bond it to the ctrtp through an amide bond. Formation of such an amide bond can be carried out using the same reaction means as described for the reaction of the hapten or its modified product with cvp at 14°C. In addition, when both the polymer latex "°1" and CFp have a carboxyl group as a functional group, the carboxyl group of the latex is chemically required by the method described below to convert it into a primary amino group. After 41 people, c
n: can also be bonded to p by an amide bond. For example, by adding a latex containing a carboxyl group 1 (butamethylenediamine strain) to the presence of a water-soluble carbosomide.
? A method of increasing primary amine groups by reacting with rimethylene diamine (e.g., Journal of Cal.
Niolngy, 64 years old, pp. 75-88 (1, 9
75) ) etc. According to the invention, for example N-ε-tertiary butoxycarbonyl nonmethyl ester or N-phthaloyl-N'-
Is it possible to remove one amino group like methyltrimethylenediamine? A thin alkylenezamine derivative can be reacted with a polymeric latex carrier having carboxyl groups. This reaction can be selected from among the amide bond-forming reactions used to bond no-butene and CWP, but it is necessary to carry out the reaction without changing the physical properties, such as the particle size of the polymer latex. be. To avoid this, it is preferable to carry out the process in a water-containing system. Soimide method is preferable. After carrying out the reaction, the amine protecting group is removed to obtain a reactive polymer latex having amine groups as reactive groups. The reactive polymer latex thus obtained was mixed with a hapten having a carboxyl group or a modified product thereof and CW.
The measuring reagent of the present invention can be obtained by reacting with a compound bound with P by the above-mentioned amide bond forming reaction. This modified aspect is explained in detail in the above-mentioned Japanese Patent Laid-Open No. 55-52945, and can be utilized in the present invention. A carboxyl group-containing water-soluble monoolefin-based polymer compound (C1l'P), which is chemically bonded to CA) no-butene or its chemical acrobatics 11 which can be obtained as described above, is As an example of a zotene-supported carrier formed by chemically bonding 1c Fir to a polymeric latex carrier having a particle size of 0.01 to about 2 microns, the following butene-supported carrier can be exemplified. . estriol-16-glucuronide acrylic acid-bonded latex, estriol-16.17-dihemisuccinate-bonded polyacrylic 2-bond latex, 17-amino-1,
3.5(10)-estratrien-3-ol bonded polyacryno bnl bonded latex, 17-amino-1,3.5(10)-estratrien-3-ol bonded vinyl methyl ether maleic anhydride copolymer bond latex, estriol-16-glucuronide-bonded vinyl methyl ether maleic anhydride copolymer-bonded latex, estriol-t6. i7-Nohemisuccinate-bonded vinyl methyl ether maleic anhydride copolymer-bonded latex, pregdensool-3-glucuronide-bonded polyacrylic acid-bonded latex, gregnanthol-3-glucuronide bound vinyl methyl ether maleic anhydride copolymer bound latex, 3α, 11β, 17α, 21-tetrahydroxypregnane-20-one-3-glucuronide-bonded polyacrylic acid-bonded latex, 3α, 11β, 17α + 21-tetrahydroxypregnane-20-one-3-glucuronide-bonded vinyl methyl Ether maleic anhydride co-bonded latex, carboxymethyl morphine-bonded polyacrylic acid-bonded latex, carboxymethyl morphine-bonded vinyl methyl ether maleic anhydride co-bonded dex, ethiocholanolone-3-hemisuccinate 4i 'i-combined polyacrylic acid-bonded latex, thyroxine-bonded polyacrylic acid-bonded latex, thyroxine-bonded vinyl methyl ether maleic anhydride co-bonded latex, metanephrine-bonded polyacrylic 1.t'12-bonded latex, metanephrine-bonded vinyl methyl ether maleic anhydride In the present invention, monoclonal antibodies that react differentially with the hapten in the (A) halogen-carrying carrier such as ``double'' are utilized. (B) prepare a carrier sensitized with a single monoclonal antibody against the hapten described above. A monoclonal antibody-producing fusion cell line is formed by appropriately selecting and combining them, and the monoclonal antibody-producing fusion cell line is used to produce the antibody.
can be obtained. According to this embodiment, a hapten or a chemically modified product thereof is combined with a substance having antigenicity, and the hapten conjugate thus endowed with antigenicity is used to inject the hapten into a suitable animal such as a mouse or a rat. , to animals such as rabbits, sheep, horses, cows, etc., by a method such as subcutaneous injection together with an adjuvant to immunize the animal (for example, an immunized mouse). Producing cells such as 胛X', lil', l'!
lIl'i! +t! Cells such as II cells, lymph node A cells, and/or peripheral blood ffl+ cells are collected, and the #ltl cells are collected.
fJ~ and a suitable cell line (U cells, for example, a mouse bone marrow lung cell line) which has self-propagating properties but does not substantially have antibody-producing ability, are fused with l1f8I cells by a method known per se. Examples of antigenic substances used to impart antigenicity to Hasithene include bovine serum albumin (BSA), rabbit serum albumin (R5Δ), ovalbumin, human serum albumin, bovine γ-globulin, and rabbit serum albumin. Examples include substances such as r-globulin, human γ-globulin, tetanus a toxin, pneumococcal polysaccharide, etc.Furthermore, the binding of halogen or its chemically modified substance with a substance having antigenicity as exemplified above can be It can be carried out by a known method (for example, Yamamura Tsui-1, edited by Kosei Ishiban, Immunochemistry, pp. 294-302, published by Rikin Shoten).
The combination of myeloma R) 11 cells and antibody-producing cells is such that each cell can fuse and proliferate while producing antibodies.The type of animal from which each cell is derived is not limited. Any combination is acceptable. There are no particular restrictions on the myeloma cell lines used, and many cell lines of animals such as mouse, rat, rabbit, and human can be used. Preferred cell lines are those that are drug resistant and in which unfused myeloma cells do not survive in the selective medium, while only fused cells survive. The most commonly used strain is the 8-azaguanidine-resistant strain #l1lltel, which lacks tA in hypoxanthine guanine phosphoribosyl transferase and can be grown on hypoxanthine aminobutyrine thymidine (IJAT) medium and exhibits femininity. have. Furthermore, it is preferable that the trill cell line used is of the "non-secreting type" [7]. For example, mouse myeloma B
opC-214 non-derived P3/X63-A (7
8UH (7'sU+), p3/×63-AClB・6・
5.3, p3/N5I-1-Ag4-1, Sp210-
Ag14, rat myeloma 210・RCY 3・Ag
1, 2, 3, etc. are preferably used. For example, normal eag/L
1 to 5x F'li' cells of the above immunized mice in a medium such as minimal basal medium (M8'M), RPMJ-1640, etc.
10” and the above mouse myeloma line cells 1iiq l~5X
10' pieces can be mixed. l'71
1. Merger; +ff4 agent has an average molecular f3.1.
(100-6.000 polyethylene glycol (pl
If ζG) is preferred, other fusion promoters can be used, such as hexapolyvinyl alcohol, viruses, etc. The use of PEG can be used at about 30-50%. HL・11 cells that can be obtained in this way 1
From the containing system, 1flll ll1J was subjected to selection treatment, antibody activity screening treatment and cloning treatment using methods known per se [7], and monoclonal antibodies against pukohapten were used to form immune mice. 1. Has the ability to suppress production and self-propagation. A unique Ivml strain can be obtained. For example, the cells that have completed cell fusion are diluted appropriately in RpJ (1-1640 medium) containing 20% fetal bovine serum, and placed in a 96-well microplate for 10' Dispense approximately 106 cells/well, add selective medium (e.g. HAT medium) to each well, and then replace the selective medium with 5 Taro C02 (incubator (37°C)).
This can be done by continuing the culture. If an 81 azaguanine-resistant strain is used as myeloma 17i11, ll1u, unfused mini0-magel ll'
・J is HAT medium" TLC kills 17, almost. Antibody-producing cells are normal cells, so in, tri, t r
o It can be grown for a long time on cultivated charcoal. (7 Therefore, after culturing 1
4i11 Month f/L that grows from about 0 to 140
II, all ρ sums are 111·1. As mentioned above, ρ! can be obtained from 1 to 1. 1 go 8111
The screening and cloning treatments for antibody activity 1 of the cell strain can be carried out by conventional methods, and for example, they can be carried out as described below in V11. After the preparation of fusion A' cells, a part of the culture medium from the 2 wells was collected and incubated with (1) stationary labeled) butene.
You can search for wells that contain the antibody of interest by visiting the state office at Kanakura 1. That is, 3HX12+! 7. After reacting the culture coat "f" with a hapten labeled with a radioisotope such as +31 I or phosphorus, etc., each reaction solution was treated with a butene-antibody (1, l',"), and the conjugate was separated by F+'. it, (
↑ By measuring the amount of discrimination, the presence and binding ability of the antibody of interest can be detected. Since there is a possibility that two or more types of fused cells are growing in each well in which the desired antibody activity is observed, cloning is performed by limiting dilution method or colony formation method using soft agar, and a monoclonal antibody against Hashiten is obtained. A production fusion cell line can be obtained (such a monoclonal antibody production fusion cell line is subject to refusal of deposit by FIKEN). To obtain a monoclonal antibody against the hapten used to form the immunized animal using the monoclonal antibody-producing cell line that can be obtained as described above, the monoclonal antibody-producing cell line is, for example, cultured in a medium,
Monoclonal antibodies can be obtained using methods known per se, such as collecting monoclonal antibodies from the culture medium, or transplanting the cell line to an animal of the same gene as the myeloma cell-derived animal, and collecting monoclonal antibodies in ascites. can. According to the former aspect, for example, monoclonal antibody-producing strain 1go6'tl1li1 is cultured in a culture solution such as RPMIIG40 medium containing 10% fetal bovine serum, and the culture solution is ammonium sulfate. A monoclonal antibody against the desired hapten can be collected by preparing 8Tj by affinity chromatography using Sepharose 4B or the like bound to an antigen. Furthermore, according to the latter case, for example, mineral oil such as pristane (2,6,10,14-tetramethylpentadecane) is intraperitoneally administered to a syngeneic animal, and then the fused cells are intraperitoneally administered. This allows the fused cells to proliferate in large quantities in υivo. As a result, the ascites that forms has a high
"Contains monoclonal antibodies of 1 degree. Monoclonal antibodies against the target hapten can be obtained from this ascites by ammonium sulfate fractionation and, if necessary, the above-mentioned affinity chromatography." Monoclonal antibodies such as those obtained in the above manner can be manually prepared and used as commercially available products. By supporting a single species on a carrier, it is possible to obtain (B) a carrier carrying a single species of monoclonal antibody against hapten. Microparticle carriers commonly used in reactions and aggregation inhibition reactions can be used, such as human, sheep, rabbit, etc. red blood cells, biological particles such as π■[bacterial cells, polymer latex, bentonite, Carriers such as collodion, cholesterol crystals, non-biological particles such as silica, kaolin, etc.) can be removed. From this] 1! In the body, the f of polymeric latex carriers can be removed.
1 for i'11. J? Listyrene latex, methyl 1/nuptasoenkov I9 limer latex, polyvinyltoluene lax, vinyltoluene/t-butylstyrene copolymer latex, styrene-methacrylate copolymer diddex, and functional groups and L7 and Cal7+? Kishir 7! -! Examples include reaction polymer latexes having an amino group or a carboxamido group (-CONJI2), and one substrate comprising the above-mentioned latex. The size of the polymer ditex body as shown in the example in Section 2 can be selected from '9'Ff:, but for example, those with an average particle size of about 0.001 to about 2 microns can be used, and Preferably, the average particle size is from about 0.05 to about 15 microns. Also, other non-biological carriers having an average particle size of about 0.01 to about 2 microns can be used in the same way as the polymeric latex phase. The method of carrying a monoclonal antibody against bald tenacin on the J't1 body as exemplified above is a conventional method of carrying an antibody against a complete antigen on the phase body, and instead of the antibody, a monoclonal antibody against bald tenn is carried on the J't1 body. It can be done in the same way except using . The method is reasonably well known and can be selected and used as appropriate in this book. As such a compatibilization method, both a manual method of adsorbing them onto a carrier and a method of chemically bonding them can be used.In the present invention, a single 9. The term "monoclonal antibody-carrying carrier" refers to all carriers in which a monoclonal antibody of a single species against bald octopus is loaded on the carrier by appropriately utilizing any of these techniques. Hereinafter, the mode will be explained in more detail. Preparation mode of monoclonal antibody-supporting carrier against hapten To sensitize a monoclonal antibody against knee hapten to a υ carrier by adsorption, a monoclonal antibody solution and a carrier are suspended.
A monoclonal antibody-supported phase complex can be easily obtained by mixing % solutions. Monoclonal antibodies often have both carboxyl groups and primary amine groups. In order to chemically bond a monoclonal antibody to a carrier, for example, 1-, +: a reactive polymer latex having the above-mentioned functional group and a monoclonal antibody may be bonded by a carbodiimide method, a carbonyldiimidazole method,
A monoclonal antibody-supporting carrier can be obtained by appropriately selecting and using one of the mixed acid anhydride method, active ester method, and the like. In addition, in order to use red blood cells as a carrier to support monoclonal antibodies, for example, a red plate is fixed with an appropriate substance such as formalin, glutaraldehyde or pyruvaldehyde, and Fuhi-fixed red blood cells are used, and if necessary, tannic acid or Monoclonal antibody-carrying blood cells can be obtained by supporting the monoclonal antibody using a condensing agent such as J, bisdiazobenzidine (B 1 B ) or glutaraldehyde. (A) A xyl group-containing water-bathable monoolefin polymer compound prepared by chemically bonding no-butene or a chemically modified product thereof, which can be prepared as described above, or a compound in which the bond is bonded to a latex carrier. Hasithene supported carrier and C
B) Immunochemical II of the present invention consisting of a combination of monoclonal antibody-supported carriers against the above-mentioned butenes.
As the constant test SEI≦, the following measurement test mulberry can be exemplified. 1. For measurement test J of Etsutroke 9, (a) A
: Estriol-16-glucuronide-conjugated polyacrylic acid-bound latex and n: anti-estriol-16-glucuronide monoclonal antibody-coupled latex. (b) A: Estriol-16. 17-ㅅhe sily-succinate-bonded polyacrylic I'i'? #, 1B combined Silatex B: Immunochemical 1111 constant reagent consisting of anti-estriol-16.37-sohemyleacusinate monoclonal antibody-supported latex, (c) A: Estriol-16
- A: Clonide-bonded vinyl methyl ether maleic anhydride (1) Polymer-bound latex and B: Anti-estriol monoclonal antibody (immunochemical assay test) A: Estriol-16 .17-dihemisuccinate ff+'r
Immunochemical assay reagent comprising a 7J'-y leic acid copolymer and B: anti-estriol monoclonal antibody-supported latex. (e) A: Immunochemical b'llll consisting of estriol-16-glucuronide-bound polyacrylic acid-bound latex and 13: anti-x triol-16-glucuronide monoclonal antibody-sensitized blood cells.
Immunochemistry consisting of anti-estriol-16.17-naccinate-conjugated polyacrylic acid and B: anti-estriol-16+'17-naccinate monoclonal antibody sensitized blood cells. (g) A: 17-7mi/-1,3,5(10)-estratrien-3-ol-conjugated polyacrylic acid-conjugated latex and B: anti-estriol-16-glucuronide monoclonal Immunization by antibody-compatible latex'O1 and target II
, 'l', llπ・i1'5, etc. 2 Pregnanthool; regular i・-('1s as t
soil, (a) A: Pregdensool-3-gluc 11 Naibishi Yoshiai polyacrylic 1'g2 bonded Radex and B:
Immunochemical assay test S1 consisting of anti-zolegnanduol-3-glucuronide monoclonal antibody Ju4-'j latex 'Malein iT'i
Copolymer and B: immunochemical measurement reagent consisting of anti-gregnathol-3-glucuronide monoclonal antibody sum J<latex; (c) A: pregnathol-3-glucuronide-bonded polyacrylic acid and B: Immunochemical assay reagent consisting of anti-zolegnanool-3-glucuronide monoclonal antibody-supported latex, 2) A: Pregdensool-3-glucuronide bond 1? Lyacrylic acid bonded latex and B: Immunochemical assay reagent consisting of anti-pregnanediol-3-glucuronide monoclonal antibody-sensitized blood cells; (E) A::76 regnanediol-3-dalcuronide bond-vinyl methyl ether maleic anhydride. An acid copolymer and B: an immunochemical assay reagent consisting of anti-regnathol-3-glucuronide monoclonal antibody-sensitized blood cells, and so on. 3. As a trial construction of 1'l-011C5, (a) A
: 3α, 11β, 17α, 21-tetrahydroxypregnane-20-one-3-glucuronide-conjugated polyacrylic acid-conjugated latex and B: anti-3α, 11β, 17α, 21-tetrahydroxyzoregnane-20-one-3- Glucuronide monoclonal antibody”11-containing latex
1: l fixed test A 5, (b) A: 3α, 11β, 17α, 2
1-tetrahydroxypregnane-20-one-3-glucuronide-bonded vinyl methyl ether outer water maleic acid copolymer and B: anti-3α, 11β, 17α, 21-detrahydroxypregnane-20-one-3-glucuronide Immunochemical assay test 4 consisting of a dextrose containing clonide monoclonal antibody Jμ
'+z N(c)noi: 3α, 11β, 17α, 21-
Tetrahydroquine pregnane-20-one-3-glucuronide-conjugated polyacrylic acid-conjugated latex and I3: anti-3α, 11β, 17α, 21-tetrahydroquine pregnane-20&-one-3-glucuronide monoclonal antibody from hemocytes. Immunochemical measurements will be wiped out (≦
, to) A: 3α, 11β, 17α, 21-tethodihydroxygregnan-20-one-3-glucuronide-bound vinyl methyl ether maleic anhydride copolymer-bound latex and B: anti-3α, 11β, 17α, 21-tetrahydro Immunochemical measurement reagents comprising latex supporting gycypregnane-20-one-3-dalcuronide monoclonal antibodies, etc. 4. As reagents for measuring morphine, (a) A: carboxymethylmorphine-bound polyacrylic acid-bound latex and B: J-carboxymethylmorphine monoclonal anti(
4,'jH! Immunochemical 8 (L place Go, (Mulberry, (C1) A: Calf) consisting of 'j' latex ?xymethylmorphine-conjugated vinyl methyl ether maleic anhydride, I (polymer and B: anti-carboxymethyl morphine) Immunochemical assay form A11 made of monoclonal antibody-supported latex;, etc. 5, 17-J (5(7)l, IL test 5, (a)
A: Ethiocholanolone-3-hemisuccinate 1-conjugated, J5 lyacrylic acid-conjugated latex and B: Anti-ethiocholanolone-3-hemisuccinate monoclonal antibody J
(b) A: Ethiocholanolone-3-to-S Zaku7ne) Binding vinyl methyl ether maleic anhydride Kyodo-Composition r1
1. Laceratex: Anti-airocholanolone-3-hemisuccinate monoclonal antibody m'tl immunochemical assay reagent consisting of blood cells, (c) A: Ethiocholanolone-3-hemisuccinate bond νj? Reacryl r (χ and B: Anti-ethiocholanolone-3-hematoclonal antibody) Immunochemical measurement reagents such as U-bearing blood cells, etc. 6 Thyroxine (T,) measurement reagents include (a) A
:Thyroxine conjugate? Immunochemical assay 4:1 consisting of lyacrylic acid-bound latex and B: anti-thyroxine monoclonal antibody-coupled latex, (B) A: thyroxine-bound vinyl methyl ether maleic anhydride 6'2 copolymer and ": anti-!J - Iroxin monoclonal antibody JII
J? Immunochemical 1411 constant assay consisting of latex, 1, (c) A:
Zyroxine-conjugated polyacrylic acid-conjugated latex and B: Immunochemical i! η1 regular exam school, etc. 7. As a constant test sample for catecholamines.
2.0) Comprising of A: metanephilin-conjugated polyacrylic acid-conjugated latex and B: anti-metanephilin monoclonal antibody-carrying latex.
b) A: Metanephilin-bound vinyl methyl ether maleic anhydride "H'5. Copolymer and B: Anti-metanephilin monoclonal antibody-supported latex, etc., immunochemical measurement reagents, etc. can be mentioned. However, the present invention The reagents are not limited to the specific examples listed above. According to the present invention, a new type of immunochemical measurement method for hapten can be provided using the above-mentioned new type of measurement reagent. According to the measurement method, (A) a carboxyl group-containing water-bathable monoolefin polymer compound to which halogen or a chemically modified product thereof is chemically bonded;
Alternatively, by using a combination of a hapten-supported carrier in which the compound is bound to a latex carrier and (B) a monoclonal antibody-supported carrier of a single type against the above-mentioned bald tenen, both of the above-mentioned CA) and (B) trial construction components are activated by the antigen in the subject. By measuring the aggregation inhibition reaction that inhibits the aggregation of the above...
Butene immunochemically i! '! 11 can be determined. The measurement consists of a qualitative 1i+111 determination to detect the presence of 7-butene in the specimen and the name of the antigen in the specimen.
1 degree j [! 1. Examples of specimens include body fluids such as urine, blood plasma, amniotic fluid, etc., or l:T, Examples include body fluid components.Hereinafter, the immunochemical measurement method of the present invention will be explained in more detail.Trace substances present in blood, urine, and other body fluids are determined by an agglutination inhibition reaction using the above-mentioned reagent.7. Although the specific measurement method is shown as an example, the general measurement method is as follows. This is an agglutination 1'-blocking reaction using an agglutination reaction system of a carrier sensitized with a monoclonal antibody against the hasithene.That is, (1) For example, one drop of a test specimen is placed on an Irr-clean slide plate. (appropriately diluted), one drop of the monoclonal antibody sensitized latex was dropped thereon, and then bald tenen-supporting latex 11 (<1) was dropped onto it.
By mixing the three components and shaking for 2 minutes, the test results are observed, and an agglutinated image is determined to be negative, and an agglutinated stain inhibition image (non-agglutinated image) is determined to be positive. (2) Put a certain amount of the sample (appropriately diluted) into a clean round-bottomed test tube, add a certain amount of monoclonal antibody-sensitized blood cell suspension to it, shake it, and mix it with water. crr='p
Bound substance suspension Binding - Add the suspension, mix by shaking, leave to stand on a stand with a mirror, and observe the image of the bottom of the tube after 2 hours. In this case, the agglutination-inhibited image (positive image) forms a sedimentation ring, and the agglutination image (negative image) has a matte appearance. Hapten sensitivity in the specimen) Currently, the test can be made by diluting the specimen appropriately and multiplying the measurement sensitivity by the highest dilution factor that gives a positive image. (, ()Cal to which a hapten or a chemically modified product thereof is chemically bonded)] of the present invention? A hydrated 11r monoolefinic polymer compound having a xyl group, or a hapten-supporting carrier in which the compound is bonded to a latex or 111 body;
1)) Immunochemical ηill determination assay for the hapten obtained from the combination of IQ-4JH monoclonal antibody-supporting carrier against the hapten and a method for immunochemical ηill determination for the hapten using the same. According to the conventional techniques using monoclonal antibodies, depending on the use of monoclonal antibodies, they bind to the corresponding antigen and form a precipitate.
Despite the fact that the color of the monoclonal antibody must be changed once, it is completely different from λ, and surprisingly, 1. l2Re (A)
And (1;) Should we add /N1 to the same number for both trials? 4
A specific reaction occurs. lji↑〈According to this book, the 4' N-i7 of a monoclonal antibody having the ability JJ and the hapten is recognized in a specific site of - in the t6-hagden molecule. Since it is an agglutination reaction based on '1 combination, it has the characteristic of achieving significantly high specificity. Furthermore, the agglutination image formed by the agglutination reaction between the above reagents (A) and (H) unexpectedly shows stronger aggregation properties than those using conventional polyclonal antibodies. It has a clear aged image and strong characteristics. Moreover, in the prior art of the use of monoclonal antibodies, there has been a tranquility in which the use of two or more different monoclonal antibodies may even be completely ineffective. It has the feature that it is free from such troubles and can be applied to a wide variety of immunological measurement methods for arbitrary amines. According to the present invention, it is possible to provide a new quiz immunochemical measurement reagent and measurement method using monoclonal antibodies having the above-mentioned characteristics, and which has significantly improved certainty, reliability, precision, and
With sensitivity and extremely high specificity, without the trouble of generating false reactions, it is possible to measure It is possible to chemically measure the bald beetles in the 01 river. The following is an example of the tree! ; Regarding Ming's analysis of numerical embodiments,
Please explain in detail. Example 1 Determination of urinary estrogen (a) Anti-estriol-16-glucuronide monoclonal antibody 4I! 11"I made of latex ((Z-1
) Estriol-16-glucuronide (Es16G
)-Production of BSA Estriol-16-glucuronide (/?, 16G
) 40m9 of N,N-dimethylformamide solution 1. . After the melting of rrteK was completed, 20.6 μl of tri-n-triamine was added to this at 4°C, and then 11.2 μl of isobutyl chlorocarbonate was added and incubated for 30 minutes.
I did it. To this, add B, SA, (bovine serum albumin) 117I'9 in advance to 2.8 m (!
Sodium hydroxide solution 1" Q/l
After adding 20πl of dimethylformamide,
The liquids kept at 8°C were mixed. Next, this was stirred at 8°C, 16.6 μl of IN sodium hydroxide solution was added after 1 hour, and after pressing for another 35 hours, Sephadex a
-25 unreacted estriol-16-glucuronide and tri? A low molecular weight reagent such as 2,-butylamine was subjected to fraction F4F. This was further subjected to M analysis (on sheath ice) and freeze-dried to obtain estriol-16-glucuronide-1) SA.The Kobel reaction of the freeze-dried powder of this antigen revealed that B571 Binding of 27 to 30 moles of siestriol-16-glucuronide was confirmed. (a-Z) Production of anti-E316G monoclonal antibody"-1
E 316 GB produced in (a), 5 A 2
-1j was injected subcutaneously into C mice (6-8 years old) at intervals of 3yJ, and 50μ7 was injected into grains during harvest. t,′,L,fy,Jl′ of mice 3 days after immunization.
l! Dispense hW, i4, collect elbow A, :++ cells, and add to Delbeco's Modified R Small Basal Medium (hereinafter referred to as /
)/ME, '+1) three times 1[,? ', f+
He is L, #, t+l 1] 7 I 37 n i, and the 2
A'S-1/ l -A g 4-1 (from N5-
1 and Tochi\zu) I
Polyethylene glycol i+ warmed to 7°C,
';ii'i (PEG-100: 4.25%, J
[At SO: 1.5 J6 containing 1 no'J/EM)
Add 1 ml of f and centrifuge for 1 minute? Rotate 1-1 slowly to perform cell fusion. 37°C glue 'Af
E “After adding 2mr of M 10 times every 30 seconds”
hs' + j min t'; in L, pellet in 209' RPlfl-1640 medium containing fetal bovine serum K11-CA'
0.2 g each was dispensed into S-t and L-t5XlO' 1121 lobe plates, and cultured in a 5% CO2 vacuum chamber. After 24 hours, half of the supernatant in the 6 wells was discarded, and HAT medium (hypoxanthine, amitubzoline, thymidine, 10
%' RpMl-1640 medium containing fetal bovine serum).
Add 1 +nl, then add half "-:li" every 3-4 days
After culturing for 2 weeks while changing the A T medium pond.
The antibody activity of the culture medium y Nijo Nerj in the wells grown with ij7 was measured. #ALJ3/C mouse thymus cells in wells with activity were added to RP containing 10% bovine fetal hematopoietin containing tin 1ii:i.
Cloning was carried out using Jfl-1640 medium using the -1 limiting dilution method to produce 10 monoclonal antibodies.
iN'i combined cells were obtained. This was cultured to maximum capacity, and the culture supernatant was extracted with E3G-1? Sepharose 4B conjugated with SA
Affinity chromatograph using [2,
Monoclonal antibodies were obtained. In addition, 2×10′ or more cells were pre-administered with pristane 0.5 rrre, B and A.
LB/C mice were injected intraperitoneally to produce ascites lung cancer. After obtaining L water, this ascites was subjected to monoclonal chromatography using Sepharose 4B conjugated with E316G-R5A. First antibody. (a-3) Production of latex with anti-E, 16G monoclonal antibody JII Anti-E 316 (y mo/rional antibody 0.2 my
5*r of glycine-rich saline solution (7+H13,2
) and 1 mp of 10% polystyrene latex was added thereto, mixed, and treated at 56°C for 30 minutes. Next, the precipitate obtained by centrifugation was diluted with glycine (□17
The heart was washed with S-; (the heart was washed with 0059σ) and suspended in 20 ml of glycine buffered saline containing BSA to produce anti-E316G monoclonal antibody-sensitized polystyrene latex [7 (b) Estriol-16-glucuronide #l'
i'fj Manufacture of polyacrylic acid-bonded latex (/1-1) Rison latex Add 260 mye of ε-tert-butyloxycarbonyllinone methyl ester to 5 mr of 109σl Ji solution of carboxyl-modified polystyrene latex and methylformamide (J) JIfF) 3 m After cooling to 0° C., 243 my of water-soluble carbodiimide [1-ethyl-a-(3-methylaminopropyl)carbodiimide hydrochloride] is added while stirring. 0℃
After stirring for 1 hour at room temperature and 3 hours at room temperature,
Leave overnight and centrifuge the molecules. The supernatant is discarded and the precipitate is washed with 50% DMF aqueous solution and then with water. Add 5 ml of ice-cold concentrated hydrochloric acid, shake occasionally and leave at 0°C for 15 minutes, then dilute to approximately twice the volume with ice water and centrifuge. The precipitate was washed until the washing solution was neutral, and then a 10% aqueous solution of triethylamine was added with 10 n·? Add 153) 4:p4 rs, = ;, :, at room temperature and repeat washing with water at 12, and wash'l + L is neutral. )
'j,, '□ξ 10 taro) It turns into a cloudy liquid; sea urchin 1.
′! Adjust the degree to obtain the desired thickness and latex. 4 items, 1, ninhydrin reaction positive. The 4vj bond of the product is expressed as 811/2 (C'H2)4. (b-2) Estriol-16-glucuronide^'
711 Go, 19 Reacryl R 1 Production (a) Estriol-16-gel curonide hexamethylene diamine derivative? Dissolve f'-estriol-16-glucuronide 93r9 and N-no-idroxicono-phosphate imide 25 +11B, l<cDM F 1.5 me, and add ncc41m9 while stirring under water cooling. 30
After 5 minutes of monobenzyloxycarponylhexamethylenediamine hydrochloride, triethylamine 0.03
A solution of ve dissolved in 1 rp/DMF was added, and stirring was continued for 2 hours under water cooling and 12 hours at room temperature. The whole was dried to dryness under reduced pressure, and the residue was subjected to Bragrate thin layer chromatography to obtain the desired Muraki compound, 82m9 (theoretical yield: 55 taro). Motoichi's silica gel thin layer chromatography showed Rf=0.42 (chloroform-methanol 5:1). (b) Dissolve 50 parts of the estriol-16-glucuronide obtained in (a) above in methanol (3πt) and add 10 rsg of radium black. , 7J% at normal pressure:
CI'; kirr11. The reaction was completed in 2 hours (7 hours).The reactor liquid was compressed under reduced pressure, and ether was added to the residue to remove nitrogen from the carbobenzyloxy group. ] 3.5 mg of the product as a powder (1↓). Dissolve this product 1oTR in 1) M F2 pr (:) with polyacrylic acid, add 1) CC4 tuna, and leave it in a room for 50 hours. . The reaction solution was subjected to one particle analysis, and the internal solution was filtered and freeze-dried to give 95 rn of estriol-16-glucuronide-bonded polyacrylic acid.
9 was obtained as a white powder. (/+-3) Estriol-16-glucuronide linkage]'Sj? Preparation of lysine latex bound to lyacrylic acid. Suspend 0.12 ml of the lysine latex produced in (b-1) above in 1 me of distilled water, and add the lysine latex produced in (b-2) above.
Estriol-16-glucuronide-bonded polyacrylic acid aqueous solution 1 rye (estriol-
16-glucuronide (equivalent to 0.311 V) and mixed, then 10 m2 of 1-ethyl-3-(3-tumethylaminopropyl)carbodiimide hydrochloride was added.
, Press-Night reaction is performed. After the completion of the reaction, the precipitate obtained by centrifugation P, a l, was washed three times with 10 rne of glycine buffered saline, and the precipitate was 0.059
0 to 1? Estriol-16-glucuronide-bound polyacrylic acid-bound latex was prepared by suspending the mixture in 30 rni of glycine-buffered saline containing SA (rabbit serum albumin). (C) Measurement of urinary nistrodan Three samples of normal female urine collected during each period of the menstrual cycle were diluted with glycine buffered saline 2 times, 3 times, 5 times, 7.5 times, 10 times, and 15 times, respectively. Dilute 20 times, 30 times, 40 times, 60 times and 80 times, and 1. Blind (0
,03+rre) onto a reaction slide plate, and then add the anti-E, 16G monoclonal antibody-supporting latex prepared in (a) above to the reaction slide, and add 121), N.
Then, 1 i + i' 4 of the estriol-16-glucuronide-bonded polyacrylic r/grape dried latex produced in (b) above is added dropwise. Mix these three components uniformly and shake for 2 minutes. With the naked eye after the movement: (:, (i'4
Statue, 7'l'jl: Gayu [stop f- toy; 1. Observe. In addition, in this example, O゛Yo measurement/+', :'&f
Since I)'l was adjusted to 2 ng/lnl, each urinary estrogen (1'll') was as shown in Table 1. Example 2 Measurement of urinary zolegnanthool ((Z) Production of Radex carrying anti-pregnanediol-3-glucuronide monoclonal antibody ((Z-1)7
'Production of regnanthool-3-glucuronide-BSA Gredananthool-3-glucuronide-BSA was produced using pregnathol-3-dalcuronide and BSA in the same manner as in Example 1 (a-1). did. (a:2) Production of anti-pregnanediol-3-glucuronide antibodies Using the pregnanediol-3-glucuronide BSA produced in (a-1) above, anti-pregnanediol 4 strains were prepared in the same manner as in Example 1 (+1-2). -3-glucuronide antibody-producing fused cells t)mi were obtained. For the monoclonal antibody, 2X10' cells were pre-incubated with silistan (15mC
Transplant into the abdominal cavity of a 13Δ7L 11/C mouse, take one pot of abdominal elbow, and dehydrate it with sulfuric acid,
The 0-talo' saturated fraction is useful as an anti-pregnanediol-3-glucuronide monoclonal antibody. (α-3) Preparation of tex having anti-gregnane diol-3-glucuronide antibody μ
- Example 1 (using a glucuronide monoclonal antibody)
α-3) In a similar manner to α-3), antigregnandiol-3-
Glucuronide monoclonal antibody carrier 4! J.I? Restyrene Radex was manufactured. (b) Gredananthol-3-glucuronide-bonded vinyl methyl ether anhydrous polymer/inr1 polymer (
Production of pVMAfΔ) (b-r) Gregnane diol-3-glucuronide lysine N#j e: Resin pregnane diol-3-glucuronide 99ffJ9
, ε-benzyloxycar is nillison methyl ester/toluenesulfonic acid 34I 140 Wow/□)//F
1. Sophenylphosphorylamide 65 and triethylamine 0 while stirring [2. Under ice cooling]
, 056)〃After adding 1, in the same manner as in Example 1 (b-2) (b-2) Production of pregnathiol-3-glucuronide bond p/7 jτ/, Mi (b-t). 3-glucuronide-lysine derivative 33nIg was contacted with f:'jI'jJLL in the same manner as in Example x(b-2), and the product was purified with pHMMA.
After adding 100 mg of MF 6 me, it was left at room temperature for 4 days. The reaction solution was treated in the same manner as in Example z (b-2) for 10 minutes to pregnate. All-containing [J O-46+I+! // me (9i(
19 Pregnanediol-3-
30 g of glucuronide-bound pvMrhΔ solution was obtained. (C) Measurement of urinary pregnane dimer 5 pregnant women's urine was diluted with dalicin saline solution 500 times and 100 times
Diluted 0 times, 2000 times, 4000 times and 8000 times, and 1 i'lJi (0,03r-/) of each diluted solution.
onto the reaction slide plate [7, and then add the anti-gledananduol-3-dalcuronide monoclonal antibody-bearing latex prepared with (σ) to this. 'I darkness', then Pregnanzio manufactured in (b) -/1. -3-glucuronide binding PVMA-I A clear solution of glycine buffered saline solution was added by 1f:・□・:J by irl'j, and the three were mixed uniformly and rocked for 2 minutes. After narcotic sleep, we observed signs of aggregation and inhibition of aggregation. However, in this example, the sensitivity of 5%% of the test was set to 5n
17/rvi, the zolegnanediol r degree of each pregnant woman's di grass was the value shown in Table 2. Example 3 Measurement of urinary 17-Of-JC5 (σ) Production of anti-3α, 11β, 17α, 21-tetrahydroxypregnan-20-one (TJIF)-3-glucuronide monoclonal antibody sensitized blood cells ( a-1) TIIF-3-glucuronide-, 13S
Production of A Using Ti1F-3-glucuronide and B, 5A,
TIIF-3-glucuronide-BSΔ was produced in the same manner as in Example 1(a-1). (a-2) Production of anti-THF-3-glucuronide monoclonal antibody Using Tl1F-'3-glucuronide-BSA produced in the above (c-t), the same as in Example 1 (α-2) Six anti-TIIF-3-glucuronide antibody Eo fusion cell lines were obtained using a method similar to that of Example 2 (a-2), and an anti-TJIF-3-glucuronide monoclonal antibody was obtained using the same method as in Example 2 (a-2). is. (α-3) Anti-T11F-3-glucuronide monoclonal antibody-sensitized hemocyte buds (suspended in formalin-fixed sheep blood cells (phosphorus lI: dehydrated (TJ chemical salt solution pH 6,4)) After adding 0.01% tannic acid solution of 41 to and reacting at 56°G for 30 minutes,
fft blood cells in phosphate buffered saline solution: After rD, 8
26 Kakeba Yoiiro. Next, a 0.0005% solution of the anti-1'11F-3-glucuronide monoclonal antibody prepared in (α-2) above was added to the mixture at 56°G for 2 hours. After the reaction is completed, phosphorus rf? Blood samples were centrifugally washed in buffered saline, and phosphorus R-wave slow-reduced food containing 0.2% NRS (original copy: infant serum) and 5% lactose was added.
Dilute 1:15 in (1 blood cell υ′ 1:1, 0.1)
Dispense nre into ampoules, then freeze and dry.
'! "Anti-T-II' F-glucuronide monoclonal antibody-sensitized blood cells were produced. (/+) Production of TRF-3-glucuronide-conjugated polyacrylic acid (b-t) 7°lf, 1' -3-glucuronide-
According to lysine derivative practical example 2 (/)-2), i'11F-3-glucuronide 5'3++y, ε-pentluoki7carnhinyllinone methyl ester/toluenesulfone RJ'
m59mp, sophenyl phosphoryl azide 331-7g
, triethylamine (1,027m) was obtained from the desired lysine ii; # conductor 57 ry (theoretical yield 7196).
Example 1 40m9 of the lysine derivative obtained in E-lyacrylic acid production procedure (b-1)
After catalytic reduction in the same manner as (-), it was reacted with 14 mg of DC and 100?5 of polyacrylic acid, post-treated, and TH
F-3-glucuronide bond boria ri') ru? l+
'l solution 15m(!(T, IIF containing L1°0.96π
y/me). (C) Three samples each of normal male urine and normal female urine with 1T-0, 110S in urine were treated with phosphorus (100x, 150x with concentrated food solution, 20041
′? , 250 times, 300 times, and 400 times, and dispense each diluted acid into a small round-bottomed H-q tube at 0.1 rnI, and then aim at (a) above. ; Suspend 1 angle of anti-TJ/F-3-glucuronide monoclonal antibody-sensitized blood cells in 0.3 ml of phosphate-relaxed I-containing solution/
Add this total amount to each and mix tV,! , 'punching, TIIF-3-glucuronide bond 11? produced by (1)) above. Reacryl's phosphoric acid solution (doi chemical salt solution 0.1
After adding mc and pushing well 17, ξ, and leaving it on a stand with a mirror for 21'I'l', it was 8 years ago, 7+
+Please observe: S lIt , IL, image (in this example, ill, !ll high sensitivity 20 n Q
Adjust to /'rne L te 4) Le (7) f 17 in each floor
-0, // CS i", :n"lIt, I, Table 3 shows the special characteristics. Example 4 Measurement of thyroxine (T4) (a) Production of latex with anti-iroxine monoclonal antibody (Q, -1) Production of thyroxine-BSA BS
, 45' Q mq to 25*r distilled water, 宕團厘7
, this (r; fil K !5 +pe (7) l)
Add 1'R'C thyroxine to NF to give 30 m9 of 1-cyclohexyl-3-
(2-mol polynyl-4-ethyl)carbosomideme 1p-)luenesulfonate was added, and the reaction was carried out overnight at room temperature. After diluting the reaction solution with distilled water, use 5
Thyroxine BSA was produced by drying. (a, -2) [F
]' I-made thyroxine U, -1.4 h in (a) above, 5
A f Jll A. Obtain an anti-Lei rogin monoclonal antibody-producing fusion cell line using the same method as in Example 1 (a, -2), and use the same method as in Example 2 (a-2) to obtain an anti- Obtained a zyroxine monoclonal antibody. (a, -3) Anti-iroxin monoclonal antibody phase" is also U+;! in latex production Jm (a-2). Dissolve the prepared anti-zyroxine monoclonal antibody 0.2h・ノ in distilled water to
'47k, 1 r of a 10% suspension of carboxyl modified latex was mixed with this, and then 10 m9 of 1-ethyl-3-(3-methylaminozolobyl) carbosomide hydrochloride was added and reacted overnight with stirring. I did this. After the reaction is complete, centrifuge i'! The precipitate obtained was washed with glycine-buffered saline, and the precipitate was mixed with glycine-moderated saline containing 008% goat serum alphamin yk10r! Ie
jl, +, p was depleted to produce an anti-thyroxine monoclonal antibody-bound latex. (b) Production of thyroxine-conjugated vinyl methyl ether maleic anhydride co-nail coalescence (PVJiAfA)-bound latex (b-1) Thyroxine-conjugated P V 71f M, 4
P L' , A
f i (/l) and thyroxine;
A white powder was obtained by drying 1;7jL. (b-2) Thyroxine binding PI/MMΔf'i
Preparation of synthetic latex The thyroxine-conjugated p
fifAi yi and implementation (3'lJ 1 (b -1)
Example 1 (1)
Thyroxine binding P)” in the same manner as 1)-3)
M, +/ΔA1″f combined latex, total latex. (') After adding 8-anilino-1-nafucrene-sulfonic acid to 4 normal male serum cases of T4 foot measurement, diluted with physiological saline 2 times and 3 times. Example 1 (c
) T4 was measured using the same procedure. The measurement sensitivity of the reagent in this example was 1°ng/Tn (!Since the VC was adjusted, the measured values were as shown in Table 4).

Claims (1)

[Scope of Claims] 1. A carboxyl group-containing water-soluble monoolefin polymer compound in which (/f) no-butene or a chemically modified product thereof is chemically bonded, or the bond is about 0.01 ~
A carrier carrying a butene chemically bonded to a polymer latex carrier having a particle size of approximately 2 microns and a carrier carrying a single type of monoclonal antibody against the above hapten are used to detect the (J) and (
B) Immunochemical z11) constant force method for butene in the above-mentioned specimen, which is characterized by measuring the agglutination inhibition reaction of both reagent components. Z (,4) A carboxyl group-containing water-soluble monoolefin polymer compound chemically bonded with a hapten or a chemically modified product thereof, or a high molecular weight compound having a particle size of about 0.01 to about 2 microns. Hapten immunity qi = chemical measurement test characterized by comprising a butene-supported 4u body chemically bonded to a molecular latex 4u body and (/?) a carrier supporting an early type of monoclonal antibody against the above hasitene. mulberry.