JPH0114540B2 - - Google Patents

Description

Detailed Description of the Invention The present invention relates to the following general formula [] A ₁ -s-s-X-s-s-A ₂ [] (wherein, either A ₁ or A ₂ is an enzyme or a thiol residue of its thiolated modified derivative, the other is an immune-related substance or a thiol residue of its thiolated modified derivative, and X represents a spacer group having 2 to 15 carbon atoms). The present invention relates to a composition for immunoassay containing a body. The present invention is based on the general formula [] (In the formula, R is a 2-benzothiazolyl group or a 2-pyridyl-N-oxide group, and X is a spacer group having 2 to 15 carbon atoms.) ,
In a nearly equimolar reaction with a compound having a thiol group, such as an enzyme, a hapten, an antigen, an immune-related substance such as a receptor, a carrier such as a protein carrier, an insoluble carrier, or a thiolated modified derivative thereof, the reaction is First, as a first step reaction, the s-s group bonded to R and the thiol group of the compound having a thiol group perform an s-s exchange reaction, and then, after the first step reaction, another thiol group is exchanged. An s-s exchange reaction is performed on the compound having the s-s group bonded to the 2-pyridyl group. 2 stage s
It has been found that this compound exhibits extremely good -s exchange reactivity. That is, it is formed by reacting a compound [ ] with a compound having a thiol group in an aqueous medium, and is schematically represented by the reaction of the following formula. (However, in the formula, A ₁ -SH and A ₂ -SH are compounds having a thiol group, and R and X are the same groups as above.) Furthermore, in place of the above 2-pyridyl group, other 4-pyridyl groups, etc. The same reaction can be achieved even if the Each compound obtained in this way is a new compound, and the compound represented as an intermediate thereof is a reactive compound useful in obtaining the corresponding final object, and furthermore, the compound shown as an intermediate thereof is a reactive compound useful in obtaining the corresponding final object. By selecting and combining compounds having a thiol group, it is possible to obtain, for example, a conjugate between an enzyme and an immune-related substance, which is useful as a labeling substance for immunoassays. It can be usefully provided as a reagent composition for immunoassay. The present invention was completed based on the above-mentioned various findings, and has the following general formula [] A ₁ -s-s-X-s-s-A ₂ [] (wherein, A ₁ , The present invention provides a composition for immunoassay containing an enzyme-immune-related component conjugate represented by A ₂ , X represents the same group as above), and provides various useful compounds and kits. First, in order to obtain the enzyme-immune-related component conjugate of the present invention represented by the general formula [] (hereinafter referred to as compound []), the above-mentioned novel compound [] or a similar compound thereof, that is, the general formula [] ] R ₁ -s-s-X-s-s-R ₂ [] (wherein, R ₁ and R ₂ represent heterocycles or aromatic ring residues exhibiting different s-s exchange reactivities) The compounds shown are used. In the compound represented by this general formula [], its s-s
The exchange reactivity is as follows. (However, s-s exchange reaction rate for glutathione at PH7.5) (However, the s-s exchange reaction rate for bovine serum albumin at PH7.5) The exchange reaction rate of the s-s group bonded to R ₁ and R ₂ in the compound represented by the general formula [] is as follows:
As mentioned above, a compound represented by the general formula [ ] may be used with the exemplified groups as appropriate, and preferably, R ₁ is a 2-benzothiazolyl group or a pyridyl-N-oxide group,
The compound represented by the above compound [], in which R ₂ is a 2-pyridyl group, has a good difference in s-s exchange reaction rate and can be effectively used. Further carbon number 2 to 15 indicated as X
The molecular spacer group is preferably an alkylene group or an alkylene group having at least one bonding group such as an amide group or an ether bonding group, and has 2 to 15 carbon atoms and is further linear or branched. It may also have a group such as a hydroxyl group, an amino group, or a carboxyl group. This spacer group can be changed as appropriate depending on the method of obtaining the compound represented by the general formula [], and in particular, an alkylene group consisting of at least 1 to 2 carbon atoms is directly bonded to the ss group. It is fine as long as it is. Furthermore, the compounds having a thiol group and the compounds represented by A ₁ -SH and A ₂ -SH used in the present invention include enzymes or thiolated modified derivatives thereof, immune-related components or thiolated modified derivatives thereof. . These compounds are not limited in any way as long as they have a thiol group, and include compounds that have a thiol group in advance and thiolated modified derivatives that have been thiolated to introduce a thiol group. In order to obtain a thiolated modified derivative, for example, an enzyme having an amino group or an immune-related component may be reacted with s-acetylmercaptosuccinic anhydride in an inert medium (Archives of Biochemistry and Biophysics).
96.605-612 (1962)), or by reacting a reactive disulfide derivative to cleave its ss group (Japanese Patent Application No. 85900/1983). In particular, when using a reactive disulfide derivative, it is sufficient to have a hydroxyl group, carboxyl group, amide group, etc. in addition to the amino group of the enzyme or immune-related component and react with the reactive group. A thiolated modified derivative is produced by cleaving the s-s group using a reducing agent such as or at a pH of about 9 to 11. Examples of this reaction are as follows. (However, in the formula, A is a residue of an enzyme-immune related component having each functional group, and X ₁ is at least s-s
( _X2 is a spacer group, R is the same group as above) Furthermore, as a thiolated modified derivative, a protein carrier such as albumin can be treated with a reducing agent to reduce the intramolecular A thiol group may be introduced by cleaving the s-s group of . Accordingly, those used in the present invention as immune-related components, enzymes or thiolated modified derivatives thereof are, for example, heroin, codeine, dihydrocodeine, morphine, estradiol,
Corticosterone, fluocinolone, barbital, nitrazepam, L-dopa, triiodothyronine, thyroxine, ergotamine, other antibiotics, poisons, pesticides, insulin, albumin, growth hormone, calcitonin, prolactin,
ACTH, PTH, glucagon, gastricin, secretin, α-phetoprotein or fragments thereof, IgG, IgA, IgM or fragments thereof, as well as antibodies against antibodies (secondary antibodies), nicotinic acetylcholine receptor, progesterone receptor, cholecalcin Immune-related components such as haptens, antigens, and receptors such as role receptors, peroxidase, catalase, cholesterol oxidase, choline oxidase, glycose oxidase, galactose oxidase, choline dehydrogenase, cholesterol dehydrogenase, glycerol dehydrogenase, lactate oxidase, lactate dehydrogenase, malate Redox enzymes such as dehydrogenase, pyruvate oxidase, L-amino acid oxidase, D-amino acid oxidase, alkaline phosphatase, phospholipase C, phospholipase D, lipoprotein lipase, lipase, glucoamylase, α-
Hydrolytic enzymes such as galactosidase, β-galactosidase, penicillin amidase, lysozyme, other transposases such as cyclodextrin glycosyltransferase, isomerase,
Enzymes such as ligase can be used. These immune-related components and enzymes are pretreated by activating their functional groups, such as amino groups, hydroxyl groups, carboxyl groups, nitrile groups, amide groups, etc., as necessary, and introducing spacers into them. When introducing these spacers, polyfunctional compounds such as ε-aminocaproic acid, ε-
Functional groups such as aminoheptanoic acid, hexamethylene diamine, dodecamethylene diamine, glutaric acid, adipic acid, glutaraldehyde, bisdiazobenzidine, hexamethylene diisocyanate, toluene diisocyanate, N,N'-ethylene bismaleimide, etc. One or more of the above may be used to react with the functional groups of enzymes or immune-related components, and for the reaction, known reactivity combinations may be selected and combined, for example, enzymes or immune-related components. For amino groups which are functional groups in related components, glutaraldehyde, hexamethylene diisocyanate, toluene diisocyanate, glutaric acid, acid azide of adipic acid, acid chloride, N-hydroxysuccinimide ester, p
- in combination with polyfunctional compounds capable of reacting with free amino groups, such as reactive derivatives of their carboxyl groups, such as active esters such as nitrophenyl esters, or by reacting amino groups with isothiocyanates to form isocyanate groups. This activated group is then combined with a polyfunctional compound such as ε-aminocaproic acid, ε-aminoheptanoic acid, hexamethylene diamine, dodecamethylene diamine, etc., and further reacted with this polyfunctional compound. The aldehyde group, amide group, and carboxyl group at the end of the spacer bonded together are further treated as functional groups in enzymes and immune-related components, and a polyfunctional compound is reacted against them to introduce the spacer. For carboxyl groups in enzymes and immune-related components, the carboxyl groups are converted into reactive derivatives such as acid azides, acid chlorides, active esters, acid imidazolides, isocyanates, etc. Combinations with polyfunctional compounds that can react with reactive derivatives of carboxyl groups such as methylene diamine and hexamethylene diisocyanate, and hydroxyl groups are activated once with cyanogen bromide, followed by hexamethylene diamine, dodecarboxylic acid, etc. A spacer may be introduced using a combination of reacting a polyfunctional compound such as methylene diamine, or using a carbodiimide reagent or Woodward reagent. Therefore, enzymes and immune-related components into which these spacers have been introduced can be reacted with the above-mentioned thiolating agent to form thiolated modified derivatives based on the functional group at the end of the spacer. Enzymes and immune-related components or thiolated modified derivatives thereof are used. Next, the compound represented by the above general formula [] is reacted with a compound having a thiol group represented by A ₁ -SH in an inert medium,
Examples of the inert medium used include water, buffer solutions, dimethylformamide, tetrahydrofuran, dioxane, and aqueous media thereof. The reaction conditions are usually 10 to 40℃.
The mixture may be stirred as necessary for about 10 minutes to 40 hours, and further carried out in a nitrogen stream. Furthermore, these may be used in equimolar proportions. The reaction products obtained in this way are recovered and purified by isoelectric precipitation, salting out, dissolution with acids, alkalis, and solvents, extraction, gel filtration, partition chromatography, and solid-liquid separation as necessary. do it. Further, depending on the purpose, the above reaction product is isolated or not isolated, and a compound having a thiol group represented by A ₂ -SH is added in an equimolar ratio in an inert medium similar to the above. In addition, usually 10
The reaction may be carried out at ~40°C for 10 minutes to 40 hours, and after the reaction is completed, it may be recovered using known isolation and purification methods. In each of the above reactions, a group bonded to the s-s group, such as a 2-benzothiazolyl group,
Since it is an s-s exchange reaction with 2-pyridyl-N-oxide group, 2-pyridyl group, etc., it liberates 2-mercaptobenzothiazole, 2-mercaptopyridine-N-oxide, 2-mercaptopyridine, etc. In particular, these liberated compounds can be measured by measuring their absorbance at their maximum absorption wavelengths or by thin layer chromatography (TLC).
The reaction can be terminated by checking the following. Further, the objective product obtained in this way has the following useful properties by selecting and combining the compounds having the thiol group. [Table] Furthermore, the compound represented by the above general formula [] can be obtained by the reaction of the following formula. [Table] The example of the compound represented by the general formula [] in the above reaction is one example, and in relation to the reaction, the thiol group may be temporarily protected with a trityl group and removed by acid treatment after the reaction. good. Furthermore, the compound [ ] of the present invention has various uses, and is used as a composition for those uses. First, the labeling substance for enzyme immunoassay in compound [] is used for qualitative and quantitative analysis of immune components such as haptens, antigens, and antibodies. It is used as an antibody, and when using corresponding immune components that immunospecifically bind to these labeled substances, such as enzyme-labeled haptens or enzyme-labeled antigens, it is difficult to specifically immunoconjugate with analytical means using competitive reaction methods. The antibody is used as the corresponding immune component, and a hapten- or antigen-containing sample such as serum, urine or saliva in the immune reaction medium is added to this to cause a competitive reaction. A solid-phase method using a solid-phase substance bound to a molecule or a two-antibody method using a second antibody is used, and when an enzyme-labeled antibody is used, the analysis method using the Sand-Deutsch method is not able to specifically detect the An antibody or a solid phase thereof is used as the immunocomponent to bind, a sample is added thereto to perform an immune reaction, and the enzyme-labeled antibody is further added thereto for analysis by the Sand-Deutsch method, Based on these various analysis methods, each necessary component, such as the corresponding antibody or its solid phase, a second antibody, and an immunoreaction medium, may be selected and combined as appropriate, and the activity of the labeled enzyme can be measured. Since it is measured as an analysis result, it is sufficient to use a reagent for measuring enzyme activity in conjunction with the enzyme activity measuring reagent. It contains the necessary components based on enzymatic reactions. For example, if oxidase is used as a label, the oxygen consumed and the hydrogen peroxide produced can be measured using an oxygen electrode or a hydrogen peroxide electrode. , peroxidase and an indicator such as 4-aminoantipyrine, N,N-dimethylaniline or phenol are used for colorimetric measurement, and preferably when a hydrolase is used as the label. It is sufficient to measure the substances decomposed and produced by enzyme action using colorimetry, etc.
Using substrates that correspond to these enzymes,
Each component necessary for the measurement is used appropriately, and therefore, when making the enzyme-labeled immunocomponents such as enzyme-labeled hapten, enzyme-labeled antigen, and enzyme-labeled antibody in enzyme-labeled immunoassay into kits, The combination may be selected as appropriate depending on the immunoassay means and the enzyme activity measurement means, and the amounts included in the composition may be changed as appropriate based on the amount of the immune component to be qualitatively and quantitatively determined in the sample. Usually
It is sufficient to have each component sufficient to measure about 0.01ng to 10μg of immune components, and 1 per test.
It is best to measure the reaction system on the order of ml, and it is usually sufficient to prepare 50 to 100 test compositions per kit. Furthermore, when using an enzyme-labeled receptor that is one type of receptor, the enzyme receptor assay may be carried out in the same manner as described above. These objects of the present invention are novel compounds that are usefully used in enzyme immunoassays,
It also binds well to its enzymes and immune-related components through an S-S exchange reaction.
It is an extremely useful compound. Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto in any way. Example 1 (1) Insulin-β-caractosidase conjugate (a) 2,2′-dithiobis(benzothiazole)
Dissolve 40g in benzene 1.5 and add 3-
Drop 8.0 g of mercaptopropionic acid and add 70
C. and stirred for 3 hours. Thereafter, the reaction solution was cooled to room temperature, concentrated under reduced pressure, and further heated at 5°C.
The crude crystals precipitated by standing overnight were collected, and the crude crystals were recrystallized with benzene to obtain 3-
16.25 g of crystals of (2'-benzothiazolyl-dithio)propionic acid were obtained (structural formula: mp163 164℃). Next, 8.13 g of this 3-(2'-benzothiazolyl-dithio)propionic acid was dissolved in 80 ml of dimethylformamide, and 3.45 g of N-hydroxysuccinimide and 6.8 g of dicyclohexylcarbodiimide were added while stirring under ice cooling.
was added and stirred for 1 hour under ice-cooling, and further stirred for 2 hours at room temperature. After that, the resulting dicyclohexyl urea was separated and the solution was added to 10 times the volume of water to collect the precipitate, which was dried under reduced pressure. 9.94 g of this dried product was repeatedly recrystallized using benzene. -(2'-Benzothiazolyl-dithio)propionic acid/N-succinimide ester 7.18g was obtained (structural formula): mp122-123℃ λ _nax 270nm (dimethylformamide: phosphate buffer (0.1M, PH7.5) =
1:9) Rf value: 0.53 (silica gel thin layer chromatography with benzene:ethyl acetate = 3:1)). In addition, dissolve 10.0 g of 2-mercaptoethylamine hydrochloride in 40 ml of water, dropwise add 4.5 ml of 30% hydrogen peroxide solution while stirring under ice cooling, stir for 30 minutes, and then add 9.0 ml of 30% hydrogen peroxide solution. was added dropwise, and then allowed to stand at room temperature for 24 hours. Next, the reaction solution was concentrated under reduced pressure at room temperature to obtain a syrup-like product, and anhydrous ethanol was added to the syrup for crystallization to obtain 10.7 g of crude crystals. This was further recrystallized using glacial acetic acid to obtain 9.77 g of 2-aminoethyl 2'-aminoethanethiol sulfonate dihydrochloride in the form of white needles (structural formula: H ₂ N-CH ₂ -CH ₂ −SO ₂ −S−CH ₂ −
_CH2 − _NH2・2HCl, mp165-160℃ (decomposition)). Then, this 2-aminoethyl 2'-aminoethanethiol sulfonate dihydrochloride 7.71
g was dissolved in 12 ml of an aqueous solution containing 3 ml of concentrated hydrochloric acid, and 3.33 g of 2-mercaptopyridine was dissolved in this.
12 ml of an ethanol solution in which was dissolved was added dropwise while stirring at room temperature. After stirring for 20 hours, ethanol was distilled off from the reaction solution under reduced pressure, chloroform was added to this to extract and remove unreacted substances, and to this solution was added a cooled aqueous solution containing 8.4 g of potassium hydroxide under ice cooling. Extract by adding chloroform, then extract twice,
These extracts were immediately diluted with concentrated hydrochloric acid, and the hydrochloric acid layers were collected, combined, and concentrated to obtain a syrup. Then, anhydrous ethanol was added to this to precipitate crude crystals.
4.91 g was obtained, which was further recrystallized from absolute ethanol to obtain 3.68 g of 2-pyridyl 2'-aminoethyl disulfide dihydrochloride (structural formula: mp150℃ (decomposition)). Furthermore, 2.0 g of 2-pyridyl 2'-aminoethyl disulfide dihydrochloride was dissolved in 20 ml of water, and PH was added with 1N potassium hydroxide aqueous solution.
After adjusting to 10, add ethyl acetate and extract.
The ethyl acetate layer was collected, washed with water, and dehydrated by adding Glauber's salt. Next, while cooling this ethyl acetate layer to -15°C, 3-(2'-benzothiazolyl-dithio) dissolved in ethyl acetate was added.
Propionic acid/N-succinimide ester
2.27 g of the solution was added dropwise with stirring, allowed to react for 90 minutes, and the precipitate was collected and dried. Further, dissolve this in chloroform and cool on ice for 5 minutes.
After washing with % aqueous sodium hydrogen carbonate solution, washing with water, and drying with sodium sulfate, the chloroform layer was concentrated. Hexane was added to this concentrated solution to obtain 2.07 g of the target product having the following structure. Structural formula: N-[2-(2'-pyridyl-dithio)ethyl]-3-(2'-benzothiazolyl-dithio)
Propionamide λmax282nm, E1% 1cm=235 (dimethylformamide: 0.1M phosphate buffer (PH7.5)=
1:9) Rf value: 0.41 (chloroform:ethyl acetate =
1:1 silica gel thin layer chromatography) 0.12 (benzene:ethyl acetate = 3:1
(Silica gel thin layer chromatography)
and then 0.1M phosphate buffer (PH
6.2) After adding 0.8ml, adjust the pH to 6.2 with 0.1M aqueous sodium hydroxide solution, add 2.5mg of s-acetylmercaptosuccinic anhydride, and adjust the pH to 6.2 with 0.1M aqueous sodium hydroxide solution. The mixture was stirred at room temperature for 30 minutes while adjusting the temperature to .0 to 6.3. Then, after the reaction,
The reaction solution was transferred to a Cephadex G-25 (manufactured by Pharmacia Fine Chemicals) column (1.5 x 75 cm, solvent: containing 0.15M NaCl).
65 through 0.01M phosphate buffer (PH7.0))
10 ml of an elution fraction (insulin fraction) having an absorption at 280 nm of 75 ml was obtained. Next, 1 ml of 0.5M hydroxylamine was added to this fraction, and the mixture was allowed to stand at 30°C for 20 minutes to cause deacetylation, and then 1 ml of this fraction was collected and passed through the above column again to give a concentration of 0.76 hydroxylamine per molecule of insulin. Fraction containing thiolated insulin with thiol group introduced into the molecule
7.5 ml was obtained (insulin content: 650 μg, [Formula] group was introduced into the amino group of the B chain lysine of insulin through an amide bond (approximately 85%)). ○C Add 50μ of the 100mM MEDTA aqueous solution to 5ml of the above thiolated insulin-containing fraction.
and N-[2-[2'-pyridyl-dithio)ethyl]-3-(2'-benzothiazolyl-dithio)
0.56 ml of dimethylformamide containing 24 μg of propionamide was added and stirred at room temperature for 30 minutes (from the absorption of 2-mercaptobenzothiazole and 2-mercaptopperidine at 311 nm and 346 nm, 98% of the thiolated insulin had reacted). , of which 95.4% reacted with the s-s group bonded to the benzothiazole group and 4.6% reacted with the s-s group bonded to the pyridyl group). ○D Next, collect 0.5ml of the above reaction solution,
Adjust the pH to 8.5 with 0.1M aqueous sodium hydroxide solution, and add 2.2mg of β-galactosidase to this.
1.0 ml of 0.01 M phosphate buffer (PH8.5) containing 0.15 M NaCl was added and stirred at room temperature for 30 minutes (2-mercaptopperidine
4.7nmole was confirmed from its absorption, and the reaction rate was
It was 96.4%. Moreover, the absorption of 2-mercaptobenzothiazole remained almost unchanged. ). This reaction solution was then diluted with 0.15M
NaCl, 0.2% bovine serum albumin (BSA),
β- A galactosidase activity fraction was obtained (47 ml to 53 ml division, activity yield 87%, ratio of bound β-galactosidase to free β-galactosidase = 92.6:7.4, simplified structural formula: (hereinafter referred to as insulin-β-galactosidase conjugate) (2) Analytical kit ○a The following [(nylon beads) - (anti-IgG antibody) - (insulin antibody)] for insulin measurement
Contains one bead with 2.0±0.11ng of insulin antibody per bead, schematically indicated by
5.0ml container. ●50μ of the above insulin-β-galactosidase conjugate/phosphate buffer (insulin content 1ng/ml). ●0.1M phosphate buffer containing 5 mg/ml of o-nitrophenyl-β-D-galactoside (0.1%
A medium for measuring β-galactosidase activity consisting of 200μ of sodium azide, 0.1% BSA, 20mM mercaptoethanol, and 10% methanol (PH6.7). ●2.5ml of 0.2M glycine buffer (PH10.4) reaction stop solution. ●Reaction medium consisting of 100μ of deinsulinized bovine serum. ●0.1% sodium azide, 0.15M NaCl,
Washing solution for reaction consisting of 0.01M phosphate buffer (PH7.2) containing 0.25% BSA and 5mM MEDTA. The above systems are combined to form a solid-phase insulin measurement kit for one test. In addition, the above [(nylon beads) - (anti-
IgG antibody)-(insulin antibody)] was obtained as follows. First, regarding insulin antibodies, 1 mg of insulin (bovine) was mixed with 0.15M NaCl.
Dissolved in 0.5 ml of 0.01M phosphate buffer (PH 7.2) containing Freund's adjuvant and homogenized to obtain 1 ml. Subcutaneously injected into guinea pigs once every two weeks. inject,
After 8 doses of sensitization, blood was collected and this was
Serum containing insulin antibodies was obtained by centrifugation at 3000 rpm for 15 minutes. This serum is
An anti-insulin IgG component, which is an immunoglobulin component, was obtained by the following purification method. Purification method 1: Add an equal amount to 50 ml of the above serum.
0.01M phosphate buffer containing 0.15M NaCl (PH
7.2) and further add saturated ammonium sulfate aqueous solution to achieve 50% saturation to obtain the precipitate, then add this precipitate to 0.01M phosphate buffer (PH7.2) containing 0.15M NaCl to make 100ml. Add ammonium sulfate to 20% saturation to remove the resulting precipitate, add ammonium sulfate to 35% saturation, centrifuge (8000 rpm, 15 minutes), and collect the IgG fraction. (yield 70%) and further this IgG fraction to 0.15M
Dissolve in 10 ml of NaCl-containing phosphate buffer (PH7.2) and charge it to a column (diameter 2 x 70 cm) packed with Sephadex G-100.
Developed with 0.01M phosphate buffer (PH7.2) to obtain the desalted pass-through fraction, and further charged the active fraction to a column (diameter 1 x 30 cm) packed with DEAE cellulose.
It was developed with 0.01M phosphate buffer (PH8.0), and the flow-through fraction was collected to obtain an anti-insulin IgG fraction (yield 59%, antibody content 1.7).
%). Purification method 2: To 2 g of Sepharose 4B (trade name) activated with cyanogen bromide, 2 ml of a 10% aqueous solution of hexamethylene diamine (PH11) was added and reacted for 60 minutes with stirring. After washing, this was mixed with 5% glutarium. Aldehyde aqueous solution (PH
8) Add 2ml to react, and then add 0.15M
It was washed with 0.01M phosphate buffer (PH7.2) containing NaCl, then 10 mg of insulin was added thereto, and [(Sepharose 4B)-NH-( _CH2 ) ₆ -N
=CH-( _CH2 ) ₃ -CH=(insulin)] Insulin-immobilized Sepharose 4B was obtained by combining the hydroxyl group of Sepharose 4B and the amino group of insulin. Next, the above-mentioned anti-insulin IgG fraction was added to this insulin-immobilized Sepharose 4B, and the mixture was stirred overnight to bind the insulin in the insulin-immobilized Sepharose 4B to its antibody component, IgG.
This was thoroughly washed with 0.01M phosphate buffer (PH7.2) containing 0.15M NaCl, and then eluted with 0.1M glycine/HCl buffer (PH2.5) to obtain affinity.
Chromatography was performed to obtain the active fraction (yield 37%, antibody purity 66%). This was used as an insulin antibody. Furthermore, regarding anti-IgG antibodies (antibodies against guinea pig IgG), rabbits were sensitized in the same manner as above using 3 mg of guinea pig IgG, and blood was collected to obtain serum. Furthermore, this serum was subjected to affinity chromatography using guinea pig IgG-immobilized Sepharose 4B obtained in the same manner as above, and was prepared using 0.1M glycine/HCl buffer (PH2.5).
An antibody against rabbit guinea pig anti-insulin IgG was obtained (yield: 37
%, purity 72%). In addition, in order to obtain antibodies against guinea pig IgG, it is sufficient to sensitize an animal of a different species than guinea pigs, and in this case, it is not limited to rabbits.
Cows, horses, etc. may also be used, and in this case, IgG antibodies from cows, horses, or guinea pigs can be obtained. Furthermore, the following procedure was carried out using nylon beads. 6.6 500 nylon beads (diameter 5 mm) were mixed with benzene 60 containing 4 g of phosphorus pentachloride and 4 g of pyridine.
ml for 2 days, and then washed 4 times with benzene to obtain imimicrolided 6.6 nylon beads, to which 50 ml of carbonate buffer (PH11) containing 1 g of hexamethylene diamine was added and stirred at room temperature for 24 hours. React, 1% heavy sodium chloride
Wash 4 times with 100ml, and add 0.1M phosphate buffer (PH) containing 2% glutaraldehyde to this.
8) Add 50ml and react at room temperature for 1 hour.
Washed with 0.1M phosphate buffer (PH8), treated again in the same manner with hexamethylene diamine and glutaraldehyde,
A spacer was introduced into the nylon beads. Next, 150 nylon beads into which this spacer had been introduced were treated with 155γ of rabbit guinea pig anti-insulin IgG antibody (anti-IgG antibody) as described above in 0.15M NaCl.
The nylon was added to 0.01M phosphate buffer (PH8.0) and reacted overnight at 5°C.
- (anti-IgG antibody)] was obtained (450-590 ng anti-IgG antibody/1 bead), and this was then combined with 10000 ng of guinea pig insulin antibody-containing serum as described above.
Double dilution solution (0.1% sodium azide, 0.15M
Contains NaCl, 0.25% BSA, 5mM EDTA
Add 15 ml (containing 350 ng of anti-insulin IgG) of a diluted solution consisting of 0.01 M phosphate buffer (PH7.2) and stir overnight at 5°C. antibody)]
Beads for insulin measurement as shown schematically were obtained. As a result of measuring the insulin antibody activity of this product, it was found that each bead had an insulin antibody activity of 2.0±0.11 ng. (The theoretical insulin antibody activity value of this product is 2.25 ng.) ○B In the measurement kit of ○B above, instead of the beads, insulin antibody (guinea pig) containing 0.4 ng of antibody, 4γ IgG A measurement kit for the two-antibody method is prepared by using normal guinea pig serum containing 4γ and anti-guinea pig IgG goat serum containing 4γ antibody (second antibody), and each system of the measurement kit in ○B. (3) Analytical method A. Insulin was measured as follows using the above-mentioned insulin solid-phase measurement kit. First, insulin 0.2ng/ml, 0.4ng/ml
ml, 0.8ng/ml, 1.6ng/ml, 3.2ng/ml,
Prepare a liquid containing insulin at each concentration of 6.4 ng/ml, 12.5 ng/ml, and 25 ng/ml,
A liquid sample containing insulin was used, and 100μ of deinsulinized bovine serum was used as the reaction medium. 100μ of this insulin-containing liquid,
The contents of the container containing 50μ of the insulin-β-galactosidase conjugate/phosphate buffer solution were injected into a 5.0ml container containing one of the beads described above, and incubated at 5°C overnight, followed by solid phase. After separating the solid phase from the solid phase and the liquid phase, and washing the solid phase with a reaction washing solution, the above medium for measuring β-galactosidase activity was added to the solid phase, and the mixture was reacted at 44°C for 2 hours. After adding the above-mentioned medium reaction stop solution to this, check the color development at 420nm.
By measuring the absorbance at a wavelength of The relationship with the amount of insulin in the sample was measured. As a result, as shown in FIG. 1, the insulin measuring kit of the present invention was useful and showed an extremely good quantitative curve. B. The method for measuring insulin using the above two-antibody assay kit is as follows. Insulin-containing liquid sample, 100 μl of deinsulinized bovine serum as reaction medium, insulin-
Pour 50μ of the β-galactosidase conjugate/phosphate buffer solution into a 5.0ml container, and add
Insulin antibody containing 0.4 ng of antibody was added and incubated overnight at 5°C, and to this, normal guinea pig serum containing 4γ IgG and anti-guinea pig IgG goat serum containing 4γ antibody (secondary antibody) were added. Incubate overnight at 5°C, then add the reaction solution to
Centrifuge at 3000 rpm for 10 minutes, collect the precipitate or supernatant, and add β
- Add medium for measuring galactosidase activity
After reacting at 44°C for 2 hours, a reaction stop solution was added thereto, and the color development was measured at 420 nm to measure the activity of each enzyme, thereby measuring the amount of insulin in the insulin-containing liquid sample. Similar good quantitative results were obtained. Example 2 (1) Insoluble carrier-insulin antibody conjugate ○A A H Sepharose 4B (1,6-diaminohexane derivative to cyanogen bromide-activated agarose: manufactured by Pharmacia Fine Chemicals) 1 g was added to 10 ml of 0.1M carbonic acid. After swelling with an aqueous sodium hydrogen solution, 10 ml of a 5% glutaraldehyde solution was added thereto, stirred at room temperature for 30 minutes, taken out, and then swollen with a 0.5 M aqueous NaCl solution.
Washed with 100ml. Furthermore, 20 ml of a 0.1 M sodium bicarbonate aqueous solution containing 0.2 g of 2-mercaptoethylamine was added to this and reacted at room temperature for 60 minutes.
Add 20 ml of phosphate buffer (PH8), add 100 mg of sodium borohydride, reduce for 60 minutes, remove, and wash with 100 ml of 0.01M phosphate buffer (PH8.5, containing 0.15M NaCl). and thiolated A H Sepharose
Got 4B. ○Ro The Journal of Immunology, 116 (6)1554
(1976), containing 1.2% insulin antibody activity.
0.5mg of Fab′ was added to 2ml of 0.1M phosphate buffer (PH
7.0), and to this, 25μ of 100mM EDTA aqueous solution and N-[2-(2'-pyridyl-dithio)ethyl]-3-(2'-benzothiazolyl-
0.5ml containing 5.5μg of dithio)propionamide
Methanol was added and the mixture was stirred at room temperature for 30 minutes, and then the reaction solution was adjusted to pH 8.5 with a 0.1M aqueous sodium hydroxide solution. ○C The above thiolated A H Sepharose
Add 20ml of 0.1M phosphate buffer (PH8.5) to 4B, and add this to the reaction solution adjusted to pH8.5 above.
0.22 ml and reacted for 60 minutes at room temperature, and then added to 0.01 M phosphate buffer (PH
7.2, containing 0.15M NaCl) and washed with 100ml.
Insoluble carrier-insulin antibody conjugate (A H
0.17 μg per 10 mg of Sepharose 4B
Fab' (conjugated with insulin antibody 2 ng) was obtained. (2) Analytical kit Instead of the insulin antibody-containing beads in the kit described in (2) Analytical kit in Example 1, 5 mg of the above-mentioned insoluble carrier was added per test.
Using the insulin antibody conjugate, a kit for solid-phase measurement of insulin is prepared using the same composition as below. (3) Analytical method In the reaction for insulin measurement described in (3) Analytical method ○A of Example 1, when separating the solid phase and liquid phase, centrifugation was performed at 3000 rpm for 10 minutes,
This precipitate was dissolved in 0.01M phosphate buffer (PH7.2,
(containing 0.15M NaCl), and the enzyme activity is measured in the same manner. Example 3 (1) Insulin antibody-β-galactosidase conjugate A. Guinea pig containing 11% insulin antibody activity
0.5mg of Fab' was dissolved in 0.5ml of 0.01M phosphate buffer (PH7.0, containing 0.15M NaCl), and 7μ of 100mM EDTA aqueous solution and 5.0μg of N-[2-(2'-pyridyl -dithio)ethyl]
Add 0.2 ml of methanol containing -3-(2'-benzothiazolyl-dithio)propionamide and stir the reaction at room temperature for 30 minutes, then PH
8.5, 0.5 ml of 0.01 M phosphate buffer (PH 8.5, 0.15 M NaCl) containing 4.9 mg of β-galactosidase was added thereto, and the reaction was further stirred for 30 minutes. Next, the reaction solution was charged to a column (2.5 x 80 cm) of Biogel P-300 (trade name), and 0.01M phosphate buffer (PH7.0, containing 0.15M NaCl) was used as the developing solvent, and insulin antibody-β- A fraction of galactosidase conjugates was obtained (free β-galactosidase content 10.8%, activated carbon content 86%). ○B Using 1 g of thiolated A H Sepharose 4B obtained in the same manner as in Example 2 (1) ○B and 5 mg of insulin antibody Fab', Example 2 was carried out.
A solid phase of antibody was obtained in the same manner as in (1) (42μ of Fab' was bound per 10mg of thiolated A H Sepharose 4B). (2) Analytical kit ○I●Solid phase of the above antibody (thiolated A
42 μg per 10 mg of H Sepharose 4B
Fab′) 10 mg. ●50 μg of the above insulin antibody-β-galactosidase conjugate (free β-galactosidase content 10.8%) as Fab′. ● Medium for measuring β-galactosidase activity: o
-0.1M phosphate buffer containing 5 mg/ml of nitrophenyl-β-D-galactoside (contains 0.1% sodium azide, 0.1% BSA, 20mM mercaptoethanol, 10% methanol)
(PH6.7) A medium for measuring β-galactosidase activity consisting of 200μ, and a reaction stop solution for the medium consisting of a 0.2M glycine buffer (PH10.4). ●Reaction medium: 0.05% sodium azide, 0.2
0.01M phosphate buffer (PH7.2) containing %BSA, 0.15M NaCl. ●Reaction washing liquid: 0.1% sodium azide,
0.15M NaCl, 0.25% BSA, 5mM
0.01M phosphate buffer containing EDAT (PH7.2). The above-mentioned systems are combined to form a kit for insulin measurement using the Sanderschschitch method for one test. (3) Analysis method Add 10 mg of the above solid phase antibody to a 5.0 ml container, add 100 μ of an insulin-containing liquid sample (containing 0.2 to 12.5 ng/ml of insulin), and add 200 μ of the reaction medium. The reaction was allowed to proceed overnight at 5°C, then an insulin antibody-β-galactosidase conjugate was added thereto, the reaction was allowed to proceed for 20 hours, and the precipitate was collected, washed with a reaction washing solution, and then added to the β-galactosidase conjugate. Add the medium for galactosidase activity measurement and react at 44℃ for 2 hours, then add the reaction stop solution, and then incubate at 420nm.
The absorbance was measured at a wavelength of m to determine the insulin content in the insulin-containing liquid sample. As a result, the kit of the present invention was a useful assay kit showing good linearity in its quantitative curve. Example 4 (1) T ₃ -β-galactosidase conjugate ○B According to Example 1 (1) ○B, 1.1 mg of T ₃ (triiodothyronine) was added to 0.1M phosphate buffer (PH
6.2) Dissolve in 1 ml, add 2.5 mg of s-acetylmercaptosuccinic anhydride and react, then charge to a Sephadex G-10 column (1.5 x 70 cm) and add 60 to 66 ml of the solution. The fractions were collected, the eluate was treated with hydroxyamine to deacetylate it, 0.7 ml of it was collected, and this was charged again to the same column as above to obtain the thiolated _T3 fraction 6.5. ml (contains 0.72 molecules of thiol group per molecule of T ₃ , thiolated T ₃ content 0.11 ng) ○B Into 5 ml of the above thiolated T ₃ solution,
63μ N-[2-(2'-pyridyl-dithio)
Add 1 ml of dimethylformamide containing ethyl]-3-(2'-benzothiazolyl-dithio)propionamide, stir the reaction at room temperature for 30 minutes, take out 0.5 ml, and add 0.1M sodium hydroxide to the reaction solution. PH8.5 in aqueous solution
After adjusting to
This reaction solution was mixed with 0.15M NaCl, 0.2% BSA,
The mixture was charged to a Sephadex G-100 column (1.5 x 80 cm) using 0.01M phosphate buffer (PH7.2) containing 0.05% sodium azide as a developing solvent to obtain a β-galactosidase activity fraction. This fraction contains free β-galactosidase.
Contains 14.9% and also thiolated _T3 and β-
It contained a T ₃ -β-galactosidase conjugate represented by the following schematic structural formula in which galactosidase was bound in a one-to-one ratio. Abbreviated structural formula: (2) Measuring kit and measuring method - Obtained as described in Example 8 below
Using a T ₃ -albumin conjugate, an antibody to T ₃ from guinea pigs was obtained by a conventional method, and an anti-IgG antibody, which is an antibody against guinea pig IgG, was used in accordance with the method described in Example 1 (2) ○ B above. Beads for T ₃ measurement (abbreviated as: [(nylon beads) - (anti-IgG antibody) - (T ₃ antibody)], each bead contains 3 ng of T ₃ antibody)
A 5.0 ml container containing one bead was obtained. ●The above T ₃ −β− containing 0.1 ng as T ₃
Galactosidase conjugate 0.2% BSA, 0.15M
100μ of 0.01MPH7.2 phosphate buffer containing NaCl
. 0.1M phosphate buffer containing 5 mg/ml of o-nitrophenyl-β-D-galactoside (containing 0.1% sodium azide, 0.1% BSA, 20mM mercaptoethanol, 10% methanol) (PH
6.7) Medium for measuring β-galactosidase activity consisting of 200μ. ●2.5ml of 0.2M glycine buffer (PH10.4) reaction stop solution. ●0.05% sodium azide, 0.2% BSA, 0.15M
Reaction medium consisting of 0.01M phosphate buffer (PH7.2) containing NaCl. ●0.1% sodium azide, 0.15M NaCl, 0.25
%BSA, 0.01M phosphate buffer (PH7.2) containing 5mM EDAT. The above systems are combined to form a kit for T ₃ measurement using the solid phase method for one test. Next, a measurement method using the above T ₃ measurement kit will be described. First, 0.1ng/ml, 0.2ng/ml, 0.4n of _T3
g/ml, 0.8ng/ml, 1.6ng/ml, 3.2ng/ml,
_T3- containing liquids at various concentrations of 6.4 ng/ml (0.2% BSA,
Contains 0.05% Sodium Azide 0.15M NaCl PH7.2
0.01M phosphate buffer) to prepare a liquid sample containing T ₃ , and using 100μ of each sample, add the above T ₃ −
Phosphate buffer containing β-galactosidase conjugate
100μ of the reaction medium was added and incubated at 5°C overnight.The solid phase was then separated and washed with a washing solution.The above medium for measuring β-galactosidase activity was added to the solid phase and incubated at 44°C for 2 hours. After the reaction was allowed to occur, a reaction stop solution was added, and the color development was measured at a wavelength of 420 nm. As a result, as shown in FIG. 2, good measurement results were obtained. Example 5 (1) Testosterone-peroxidase conjugate ○A 20 mg of testosterone and 40 mg of hexamethylene diamine were dissolved in 1 ml of potassium hydroxide-methanol solution, stirred at room temperature for 150 minutes, the solvent was distilled off, and water was added thereto. and then centrifuging the mixture to collect the precipitate.
Dry to give 6-aminohexamethyleneiminotestosterone (benzene: methanol = 9:
24 mg of Rf=0.31 was obtained by silica gel thin layer chromatography according to No. 1. Collect 0.66mg of this and add it to 0.5ml of dimethylformamide.
Dissolve in 1 ml of 0.1M phosphate buffer (PH6.2)
was added, the pH was further adjusted to 6.2 with hydrochloric acid, 2.4 mg of s-acetyl mercaptosuccinic anhydride was added thereto, and the mixture was reacted for 30 minutes. Hydroxyamine was further added to deacetylate, and after the reaction, acetic acid was added. ethyl 20ml
was added, washed 8 times with 20 ml of water, ethyl acetate was distilled off, and the residue was dissolved in 1 ml of methanol (the rate of introduction of thiol groups per molecule was
0.65 molecules). ○ 6.7 mg of peroxidase (horseradish),
Dissolved in 1 ml of 0.1 M phosphate buffer at pH 6.2, added 0.1 mg of s-acetylmercaptosuccinic anhalide, reacted in the same manner as above, and further deacetylated with hydroxylamine to give 5.63 mg. A fraction of thiolated peroxidase was obtained (the rate of introduction of thiol groups per molecule was 1.07 molecules). ○C 74μ of the above thiolated testosterone
In 2 ml of 0.1 M phosphate buffer (PH 7.0) containing 20% methanol containing
200 .mu. of a methanol solution containing 45 .mu.g of ethyl]-3-(2'-benzothiazolyl-dithio)propionamide was added and reacted at room temperature for 30 minutes. Next, take 1 ml of the sample, add 0.5 ml of 0.1 M phosphate buffer (PH 8.5) containing 1.6 mg of the above thiolated peroxidase, and further adjust the pH with an aqueous sodium hydroxide solution.
8.5 and allowed to react at room temperature. Then, the reaction solution was charged to a Sephadex G-50 column (1.5 x 75 cm) to obtain its active fraction, and a testosterone-peroxidase conjugate was obtained (active Yield 74%, free peroxidase 17%, simplified structural formula: Example 6 (1) Insulin-β-galactosidase conjugate ○A 5.05 g of 2,2'-dithiobis(pyridine-N-oxade) was dissolved in 200 ml of chloroform, and 2.55 g of 3-mercaptopropionic acid was dissolved in this.
g was added dropwise, stirred at 40°C for 1 hour, and then the reaction solution was cooled to room temperature to collect a precipitate. This precipitate was further recrystallized from chloroform to give 3-(2'-pyridyl- 3.72 g of N-oxide-dithio)propionic acid was obtained (Rf
Value: 0.63 (n-butanol:acetic acid:water=4:
1:1 silica gel thin layer chromatography) In addition, 2.0 g of 2-pyridyl 2'-aminoethyl disulfide dihydrochloride obtained in the same manner as in Example 1 (1) A was added to 20 ml of water. Dissolve and adjust pH to 10 with 1N aqueous sodium hydroxide solution under ice-cooling.
The chloroform layer was extracted with chloroform, washed with water, dried with sodium sulfate added thereto, and further concentrated under reduced pressure. Furthermore, the above 3-(2'-pyridyl-N-
Oxide-dithio)propionic acid 1.19g
was dissolved in 50 ml of tetrahydrofuran, and 2 ml of a tetrahydrofuran solution containing 1.06 g of dicyclohexylcarbodiimide was added dropwise to this solution while stirring under ice cooling. After 20 minutes, this solution and the above concentrated solution were combined with stirring under ice cooling, stirred for 1 hour, and then continued to stir at room temperature for 3 hours. Dicyclohexylurea produced after the reaction was removed, water was added to the solution, chloroform was further added, and the chloroform extract was collected. Furthermore, this chloroform layer was added to 5%
After washing with hydrochloric acid, a 5% aqueous sodium bicarbonate solution, and water in this order, it was dried by adding Glauber's salt, and chloroform was distilled off to obtain 1.46 g (purity 91%) of the target product having the following structure. Structural formula: N-[2-(2'-pyridyl-dithio)ethyl]-3-(2'-pyridyl-N-oxide-
dithio)propionamide Rf value: 0.55 (n-butanol:pyridine:
Silica gel thin layer chromatography using an upper layer solution of acetic acid:water = 10:3:0.1:11) ○B 0.01M containing 590 μg of thiolated insulin obtained in the same manner as in Example 1 (1) ○B.
Phosphate buffer (PH7.0, containing 0.15M NaCl) 5
60μ and 38μg of 100mM EDTA aqueous solution in ml
N-[2-(2'-pyridyl-dithio)ethyl]-3-(2'-pyridyl-N-oxide-
1 ml of dimethylformamide containing dithio)propionamide was added and reacted with stirring for 30 minutes at room temperature (as a result, 94% of the thiol groups of thiolated insulin were reacted and introduced, and 97% of them were converted to pyridyl- N
- reacted with the s-s group bonded to the oxide group). Then add 0.1N to this
The pH was adjusted to 8.5 by adding an aqueous sodium hydroxide solution. ○C Take 0.3ml of the above reaction solution and add 2.4ml to it.
0.5ml of 0.01M phosphate buffer (PH8.5, containing 0.15M NaCl) containing mg β-galactosidase
was added and allowed to react at room temperature for 60 minutes, and then the reaction solution was charged to a Sephadex G-100 column and diluted with 0.01M phosphate buffer (PH7.2) containing 0.15M NaCl, 0.2% BSA, and 0.05% sodium azide. The active fraction of the insulin-β-galactosidase conjugate was obtained by developing and eluating the active fraction (activity yield: 83%, free β-galactosidase content: 14%). In addition, the schematic structural formula of the insulin-β-galactosidase conjugate of the present compound is shown in Example 1.
It is the same as that shown in Example 1, and thus can also be used for analysis as in Example 1. Example 7 (1) 1-34h-PTH-β-galactosidase conjugate ○A 2 mg of 1-34h-PTH (Proceeding of the
National Academy of Sciences of the
USA, 71 (2) 384-388 (1974), human parathyroid hormone consisting of an amino acid sequence of 1 to 34 (hereinafter simply referred to as PTH) was added to 0.01M veronal buffer (PH8.0). 0.2
To this was added 0.1 ml of dimethylformamide containing 15 μg of 100 mM EDTA aqueous solution and 260 μg of s-acetylmercaptosuccinic anhydride, and reacted at room temperature for 60 minutes. After the reaction, a Sephadex G-25 column (1.0 x 50 cm) was added. charge to that
Obtain a PTH-containing fraction and then add 0.2
ml of 0.5M hydroxylamine was added for deacetylation, and the mixture was charged to the same column as above to obtain a fraction containing thiolated PTH (the number of thiol groups per molecule of PTH was 0.33). ○B Add N-[2-
(2'-pyridyl-dithio)ethyl]-3-
Dimethylformamide containing 5.2 μg of (2′-benzothiazolyl-dithio)propionamide
0.1 ml was added and reacted at room temperature for 30 minutes. ○C Next, take 0.3 ml of the solution after the above reaction, add 2 ml of 0.1 M phosphate buffer (PH8.5) containing 2.5 mg of β-galactosidase, and stir the reaction at room temperature for 60 minutes. Next, this reaction solution was charged to a Sephadex G-200 column (1.5 x 75 cm) and eluted with 0.01M phosphate buffer (PH7.2, containing 0.15M NaCl) to collect the β-galactosidase active fraction. A PTH-β-galactosidase conjugate was obtained (activity yield 87%, free β-galactosidase content 35%). Example 8 (1) Example 2 of insoluble carrier-insulin antibody binding
Application to ○A A H Sepharose 4B (1 g) was swollen with 10 ml of 0.1 M sodium bicarbonate aqueous solution, then 5% glutaraldehyde (10 ml) was added and reacted with stirring at room temperature for 30 minutes.
Washed with 100 ml of 0.5M NaCl aqueous solution. This then contains 0.2g of mercaptoethylamine.
20 ml of 0.1M sodium hydrogen carbonate aqueous solution was added and reacted at room temperature for 60 minutes. After the reaction, collect and wash with 0.1M phosphate buffer (PH8.0).
Further add 20ml of 0.1M phosphate buffer (PH
8.0), and 100 mg of sodium borohydride was added to this and reduced for 60 minutes. After the reaction, this was taken out and 0.01M containing 0.15M NaCl was added.
Thiolated A H Sepharose 4B was obtained by washing with 100 ml of phosphate buffer (PH8.5). ○B Guinea pig containing 11% insulin antibody activity
Dissolve 0.5 mg of Fab′ in 2 ml of 0.01 M phosphate buffer (PH7.0) containing 0.15 M NaCl, and add
0.2 ml of a dimethylformamide solution containing 5.3 μg of 1-(2'-benzothiazolyl-dithio)-6-(2'-pyridyl-dithio)hexane
was added and reacted with stirring for 30 minutes. After the reaction, the reaction solution was diluted with 0.1N aqueous sodium hydroxide solution.
Adjusted to PH8.5. ○C Next, the above thiolated A H
Sepharose 4B with 20ml of 0.15M NaCl
Add 0.01M phosphate buffer (PH8.5) to suspend, add this to 0.2ml of the above reaction solution adjusted to PH8.5, stir the reaction at room temperature for 60 minutes, collect after the reaction, 0.15 M Contains NaCl
Wash with 100ml of 0.01M phosphate buffer (PH7.2) and add 0.38μg per 10mg of A H Sepharose 4B.
An inert carrier-insulin antibody conjugate to which Fab' was bound was obtained. (Also, this product is used in the same manner as in Example 2.) The above 1-(2'-benzothiazolyl-dithio)-6-(2'-pyridyl-dithio)hexane is as follows: You can get it like this. 2,2'-dithiobis(benzothiazole)
20mg was dissolved in 40% ethanol-containing 0.1M phosphate buffer (PH6.4, 1mM EDTA) 3, and 397mg of 2,2'-dithiobis(pyridine) was dissolved in the same buffer 1 as above.
The same buffer solution 1 as above in which 9.0 mg of 1,6-dimercaptohexane was dissolved was added with stirring at room temperature, and the mixture was allowed to react for 60 minutes. After the reaction, ethanol was distilled off under reduced pressure, and the mixture was extracted three times with chloroform. The chloroform layers were collected and combined. Glauber's salt was added thereto, dried, and concentrated. Furthermore, this concentrated solution was charged to a silica gel packed column (diameter 1.0 x 40 cm),
It was eluted with an elution solvent of benzene:ethyl acetate = 20:1, and the fraction containing the target product was collected, and this was dried under reduced pressure to obtain 1-
(2′-Benzothiazolyl-dithio)6-(2′-
16.5 mg of pyridyl-dithio)hexane was obtained. λmax of this product = 279nm (dimethylformamide: 0.1M phosphate buffer (PH7.5) = 1:
9), Rf = 0.51 (benzene: ethyl acetate = 10:
Silica gel thin layer chromatography according to No. 1) and its structural formula is as follows. Also, in place of the above 1,6-dimercaptohexane, 1,2-dimercaptoethane,
1,4-dimercaptobutane, 1,8-dimercaptooctane, 1,9-dimercaptononane, 1,10-dimercaptodecane, di(2
-mercaptoethyl) ether, dithioerythritol, and dithiothreitol in equimolar amounts, the following compounds were obtained. In addition, all of these compounds have the above-mentioned 1-(2'-benzothiazolyl-dithio)-6-
It exhibits s-s exchange reactivity like (2'-pyridyl-dithio)hexane, and is used in the same manner as above. Example 9 (1) Insulin antibody-enzyme conjugate A guinea pig containing 11% insulin antibody activity
Fab′0.5mg, 0.5ml of 0.15M NaCl containing 0.01M
Dissolved in phosphate buffer (PH0.7),
5.3 μg of 1-(2'-benzothiazolyl-dithio)
Add 0.2 ml of a dimethylformamide solution containing 6-(2'-pyridyl-dithio)hexane and stir the reaction at room temperature for 30 minutes. After the reaction, the pH of the solution
was adjusted to 8.5, and 0.5 ml of 0.15 M NaCl-containing 0.01 M phosphate buffer (PH 8.5) containing 4.8 mg of β-galactosidase was added thereto, followed by a stirring reaction at room temperature for 30 minutes. After the reaction, the reaction solution was charged to a Sephadex G-200 packed column (1.5 x 80 cm) and developed and eluted with 0.1M phosphate buffer (PH7.2) containing 0.15M NaCl, 0.2% BSA, and 0.05% sodium azide. 45 to 52 ml of the fraction was collected to obtain an active fraction of insulin antibody-β-galactosidase conjugate (free β-galactosidase content 7.5%, activity yield 82%).

[Brief explanation of drawings]

FIG. 1 shows an insulin quantification curve of an insulin-containing liquid sample obtained using the insulin-β-galactosidase conjugate in Example 1, and FIG. 2 shows an insulin quantification curve obtained using the T ₃ -β-galactosidase conjugate in Example 4. The _T3 quantitative curve of the _T3 -containing liquid sample is shown.

Claims (1)

[Claims] 1 The following general formula [] A ₁ -S-S-X-S-S-A ₂ [] (However, in the formula, either A ₁ or A ₂ is an enzyme or its thiol. one is a thiol residue of a thiolated modified derivative, the other is a thiol residue of an immune-related substance or its thiolated modified derivative, and X represents a spacer group with 2 to 15 carbon atoms). A composition for immunoassay. 2. The immunoassay composition according to claim 1, wherein the immune-related substance is a hapten, an antigen, or a receptor.