JPS6035622B2 - Enhancement medium for β-galactosidase activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides an enzyme immunoassay using an 8-galactosidase-conjugated immunocomponent.
The present invention relates to a medium for immunoreaction measurement in skin assay (hereinafter abbreviated as "EIA"), which has enhanced 8-galactosidase activity.

Conventionally, in an immune reaction measurement system in EIA, the activity of the enzyme label has been used for measurement.

However, in order to improve the activity in EIA and make the measurement of the reaction system sensitive, various highly active enzymes were searched for, but the stability of the enzyme itself - the enzyme bond - was investigated. When used as an immune component, there were various problems such as stability of the enzyme portion and deterioration of immune activity. Furthermore, it has been known that heavy metal ions, surfactants, and alcohols have been used to influence the activity of 8-galactosidase [Biochemical
and Biophysics Research
hComm skin cations Vol 18, ground. 5
16, A 725-734 (1965)].

However, the purpose of using these heavy metal ions, surfactants, and alcohols for 3-galactosidase is to indicate the properties of the enzyme itself, but only to indicate the properties of the enzyme's activity enhancement, deterioration, and inactivation. It was something.
However, the effects on 8-galactosidase activity due to the addition of heavy metal ions, surfactants, and alcohol, which indicate the properties of the enzyme itself, merely indicate the activity of the enzyme itself; 8-galactosidase and immune components The effect of addition of heavy metal ions, surfactants, and alcohol on the activity of modified compounds of 8-galactosidase, which is a bonded immune component, was completely unknown. Furthermore, the 8-galactosidase-conjugated immunocomponent and other reagents used in the immune reaction in EIA are used in EIA with a significant amount, and the addition of alcohol, etc. in the reaction system of such a amount may cause The effects were completely unknown. In particular, there have been various problems such as the adverse effects of heavy metal ions and surfactants on immune reactions, such as the problem of turbidity caused by the formation of complex salts, and the adverse effects on immune components caused by the addition of alcohols. The present inventors have developed an EIA method using an 8-galactosidase-conjugated immunocomponent comprising 8-galactosidase as an enzyme label.
As a result of various studies to enhance the 8-galactosidase activity of 3-galactosidase binding, an immune component, and to sensitively measure the immune response system, it was surprisingly found that some alcohols and ketones It has been found that B-galactosidase binding - 3-galactosidase activity of the immune component can be successfully enhanced without adversely affecting the immune response. When used in an amount of 5 to 30%, the activity is significantly enhanced, and when combined with a compound having a thiol group, the range of hydrophilic alcohols and ketones used is 10 to 15% of the total amount. I learned that the activity can be further enhanced, although the amount is limited to %.

The medium thus obtained is a useful activity-enhancing medium that can be well used when 8-galactosidase is used as an enzyme label in measurement systems such as detection and quantification of constituents of biological components in EIA. I learned that. The present invention was completed based on the above findings, and
8-Galactosidase binding-EIA using immunocomponents
As an immune reaction measurement system medium, it contains 10 to 15% of the total amount of one or more compounds selected from the group consisting of hydrophilic alcohols and ketones, and a compound containing a thiol group. A medium for enhancing the 3-galactosidase activity of an 8-galactosidase-binding immune component in EIA using a system that uses a system that The present invention provides a medium for enhancing 8-galactosidase activity as measured by EIA.

First, the immune reaction according to the present invention refers to a compound having an immunological effect, which is usually called an antigen, antibody, or papten.

These immune reactions include, for example, insulin, calcitonin, albumin, growth hormone, prolactin, and
CT day, parathyroid hormone, glucagon, gastrin,
secretin, /tankreozycin, cholestikinin,
Isoenzyme, LH-RH, y-globulin, 1gG,
1gY, 1gA, estrogen, androgen, thyroxine, triiodothyronine, angiotensin,
Prostaglanden, tecolamine, glurocorticoid, andosterone, ATP, morphine, code 1
In the hapten, ergotamine, L-do/g, pheno/rubital, diaze/gum, oxazepam, ampicillin, amoxicillin, chlorbenicillin, cyclacillin, cephaloglycine, cefazoline, cephalothin, creatinine, creatine, etc. If necessary, an amino group, hydroxyl group, carboxyl group, or thiol group may be introduced by a known method. Next, in order to obtain the 8-galactosidase-conjugated immunocomponent used in the present invention, a polyfunctional binding agent used for binding 8-galactosidase and the above-mentioned immunoreaction, such as water-soluble carbodiimide, is used. , p.p'-difluoro m.m'-dinitrophenyl sulfon, glutaraldehyde, NN-0-phenylene dimaleimide, azirylcarboxylic acid derivative, m-maleimidobenzole, N-hydroxysuccinimide ester, W-amino acid derivative, maleimide substitution. This may be carried out using a fatty acid/succinimide ester conductor or the like.

These components are reacted by an appropriate reaction method, such as first reacting with an immune reaction, 8-galactosidase, and a binding agent simultaneously, or reacting an immune reaction with a binding agent and then reacting with 8-galactosidase. ,
In this reaction, free groups that do not participate in the reaction may be protected with a protecting group that can be easily removed by ordinary oxidative water decomposition using trifluoroacetic acid or hydrogen fluoride or reductive decomposition using palladium-carbon. , e.g. methyl group,
Ethyl group, benzyl group, trityl group, tosyl group, benzyloxycarbonyl group, 2,2,2-trifluoro-1
In general, protecting groups such as rt-butyloxycarbonylaminoethyl group are mentioned, and the carboxyl group involved in the reaction can be formed by acid halide, active esterification, etc. In this active ester, ordinary peptide Active esters used in the synthesis, such as cyanomethyl ester, thiophenyl ester, p-nitrothiophenyl ester, p-methanesulfonylphenyl ester, thiodyl ester, p-nitrophenyl ester,
2,4-dinitrophenyl ester, 2,4,5-trichlorophenyl ester, 2,4,6-trichlorophenyl ester, pentachlorophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalic acid imide ester , 8-hydroxyquinoline ester, N-hydroxypiberidine ester, etc., and if necessary, NN-diisopropylcarbodiimide,
NN'-dicyclohexylcarboimide, N-ethyl-N'-dimethylaminopropyl-carbodiimide, NN
The carboxyl group is converted into an active ester using a condensing agent such as -carbonyl-bisimidazole, isoxazolium salt, diphenyl phosphoric acid amide, diethyl phosphoric acid cyanide, etc. Furthermore, a medium is used in the above bonding reaction, and this medium may be any inert solvent, such as water, acidic aqueous solution, basic aqueous solution, methanol, ethanol, isopropyl alcohol, acetone, tetrahydrofuran, Dimethylformamide, dioxane, dimethyl sulfoxide, ethyl acetate, benzene, toluene, etc. are selectively used alone or in combination, and the reaction temperature is preferably room temperature, and the ratio of their use is such that the desired 8-galactosidase bond is achieved. - It may be used in the necessary molar ratio depending on the immune component. For example, if the purpose is to bind 1 mole of insulin to 1 mole of B-galactosidase, first 1 to 2 moles of the binder to 1 mole of insulin can be used. 1 mole of 8-galactosidase may be added immediately or after the reaction, and the reaction may be carried out by adding 1 mole of 8-galactosidase.If the reaction is to be carried out in two or more steps, it may be filtered in an oven as necessary, collected,
The B-galactosidase-bound immune component obtained by these reactions may be washed and subjected to color chromatography, membrane filtration, gel furnace, etc. It may be purified using the following purification means. Furthermore, the 8-galactosidase binding-immune component is not limited to those mentioned above. Furthermore, the hydrophilic alcohols used in the present invention may be hydrophilic alcohols having 1 carbon number.
-3 alcohols are mentioned, and among these alcohols, mono- to trihydric alcohols are mentioned,
Examples include methanol, ethanol, propanol, propylene glycol, glycerin, etc., with methanol and ethanol being preferred as compounds that significantly enhance the effect.

Furthermore, examples of hydrophilic ketones include ketones such as acetone and methyl ethyl ketone. moreover,
These hydrophilic alcohols and ketones may be used as they are, or in an aqueous medium such as water, p
It may be added with H6.8 phosphate buffer,
The point is that the amount of these hydrophilic alcohols and ketones used should be 10 to 15% of the total amount.

Examples of compounds having a thiol group that can be used include glutathione, mercaptoethane, dithiothreitol, thiosalicylic acid, thiamine hydrochloride, benicillamine, thioproline, thioctic acid, and cysteine, preferably glutathione, mercaptoethane, and dithiothreitol alone. Alternatively, they may be mixed and added. At this time, the amount of the compound having a thiol group to be used is usually 1 to 3
The amount is about 0 mmol, preferably about 10 to 25 mmol. Furthermore, these systems may be prepared by adding, for example, a pH 6.8 phosphate buffer. Next, the target 8-galactosidase-binding immune component is added to the 8-galactosidase activity enhancing medium obtained as described above, either as it is or added to a buffer, and after the desired immune reaction is completed. The enzyme activity of 8-galactosidase is measured, for example, aryl or alkyl-8-D-galactoside, specifically o-nitrophenyl-B-D-galactoside, p-nitrophenyl-8-galactoside, 6-galactosidase. -Hydroxyfluoran-D-galactoside, etc., and then an enzymatic reaction of about 37 to 45 qo for about 30 to 60 minutes, and then the reaction is stopped and the resulting decomposition products, such as o-nitrophenol, p-nitrophenyl, D −
Galactose and the like may be measured by known means, such as colorimetry or reducing sugar quantification.

The 8-galactosidase activity enhancing medium obtained as described above is useful in EIA, and for example, by using the medium of the present invention in that case, a high activity of 2 to 3 times that of conventional B-galactosidase can be obtained. It will be shown,
This greatly improves the confirmation and measurement of 8-galactosidase activity.

Next, to specifically describe the present invention, comparative data will be given and examples will be shown.
There are no limitations on the temperature, the means for measuring the enzymatic reaction, etc.

Example 1 [11 Enzyme activity measurement method (principle) o Nitrophenyl-81-galactoside L-millet toxin Umirenmica scent-immune component 0-nitrophenol-D-galactose↓buffer (pHIO.4 ) Measure the yellow color of 0-nitrophenol in a max 42 melon dish.

(Method) . 0.1M phosphate buffer, pH 6.8 0.
2 kites [(lmMM and 12, 0.1M NaCI, 0.1%
Bovine serum alpmin, containing 0.1% NaM3)・10 females/
Synthetic (buffer) o Nitrophenyl-8-D-galactoside 50 Enhancement medium for mu-8-galactosidase activity or control medium (target amount used for total volume) 37-490, reacted for 50 minutes in a water bath. rear,
The reaction was terminated by adding 0.1M glycine-sodium carbonate buffer (pHIO.4) 2.5% as a reaction terminator, and the absorbance was measured at 42 m to determine the enzyme activity (relative activity). I asked for it.

} In the above method for the P-galactosidase activity enhancing medium using 8-(8-galactosidase)-dehydroxyseryl-glycyl-insulin,
8-(B-galactosidase)-dehydroxyseryl-glycyl-insulin synthesized as described below was used as a compound having an 8-galactosidase-binding-immune component, and methanol and ethanol were used as enhancing media for 8-galactosidase activity. , acetone were used, respectively.

Medium used and amount used Activity (%) No additives
100 methanol 5%
191 Methanol 10% 2
09 Methanol 15% 214 Ethanol 5% 142 Ethanol 1
0% ・68 kyu/ru -5%
171 Acetone 5%
128 Acetone 10% 14
2 Acetone 15% 134 As a result, when using 8-(B-galactosidase)-dehydroxyseryl-glycyl-insulin, the amount of each medium to be used is about 10 to 15% of the total amount, so that no additives can be used. In the case of methanol compared to 209 ~
This was seen from an increase in activity of 214%, 168-171% for ethanol, and 134-142% for acetone.

Thus, it has been found that methanol, ethanol, and acetone are preferable. Moreover, this 8-(8-galactosidase)-dehydroxyserylglycyl-insulin was obtained as follows.

N-Tritylazilylglycine benzyl ester (13th Veptide Symposium, p. 30, 1973) 960-9
was dissolved in ethyl acetate, treated with a small amount of palladium-carbon as a catalyst, treated in a hydrogen stream at room temperature for 30 minutes to undergo catalytic reduction, filtered in an oven, and after distilling off ethyl acetate, petroleum ether was dissolved. In addition, it was washed, dried under reduced pressure, and prepared using N-tritylazilylglycine 680-9 (a developing solvent of chloroform:methanol:acetic acid = 95:5:1) and a silica gel plate called Mercquart 5721. The TLC Rf value was 0.19.

In addition, a max of 26 melons (in ethanol) was obtained. 9 of this N-tritylazilylglycine 208 was separated, and further 9 of 74.3 of N-hydroxysuccinimide was weighed, and these were dissolved in 5 M of tetrahydrofuran. ℃ cooling, the 6 powders were reacted, kept at 5℃ overnight, and then the lower molten part (dicyclohexylurea) was removed from the reaction solution in a suction oven, and then the tetrahydride was removed. Furan was distilled off under reduced pressure, and N-tritylazilylglycine succinimide ester 250:9 (TLC using a developing solvent of chloroform:methanol:acetic acid = 95:5:1 and a silica gel plate) had an Rf value of 0.60. ) was obtained. Next, 9 of 16.7 of this N-tritylazilylglycine succinimide ester and 9 of 150 of insulin were added to 0.1N of sodium hydroxide solution.
5 [and dimethylformamide 1], reacted at room temperature for 120 minutes, then adjusted to pH 5.3 to 5.4 with hydrochloric acid, and collected the resulting precipitate. was freeze-dried to obtain N-tritylazilylglycyl-insulin 106 powder [white powder, Rf value of TLC using a developing solvent of butanol:pyridine:acetic acid:water = 15:10:3:12 and a silica gel plate. 0.60 (the TLC Rf value of insulin using the same solvent is 0.47), and the input max 2
It was 77 nm. Furthermore, this N-tritylazilylglycyruinsulin 1F was dinitrophenylated in a conventional manner, and hydrolyzed with carp hydrochloric acid for 1,060 hours for 1 hour, and the dinitrophenylated amino acids were quantified.
Since dinitrophenylated glycine of makum M and dinitrophenylated phenylalanine of 0.97 mt were obtained, and no dinitrophenylated lysine was detected, N It is clear that a -tritylazilylglycyl group has been introduced. ] was obtained. Furthermore, 9 of this N-tritylazilylglycyl insulin 11.3 is 0.1
In addition to anhydrous methanol, add 0.5 ml of trifluoroacetic acid at 0°C and react for 15 minutes, then remove methanol and trifluoroacetic acid under reduced pressure, wash with anhydrous ether, and add sodium hydroxide. Dry under reduced pressure in a desiccator and add azilylglycylinsulin 10.
．． 3c [white powder, Rf value of TLC using a developing solvent of butanol:pyridine:acetic acid:water=15:10:3:12 and a silica gel plate is 0.15,
It was max 27m. See you~zmejmitte
As a result of quantification according to l-FoRh (19) 1059'69, 0.92 mA of aziridine was detected for 1 mA of insulin. ] was obtained. Next, 0.256 parts of this azilylglycyl-insulin 9 was added to 0.4 parts of 0.1 M phosphate buffer (pH 7.5), and then commercially available 8-
Galactosidase (manufactured by Sigma) 16m9 (700U/
Add 9) and react for 400 minutes for 1 hour.
After the reaction was completed, this solution was charged to a column (1 x 50 cells) packed with Sephadex G-200, and 0.02M phosphate buffer (pH 7.5), 0.19M NaCI and
．． One fraction was eluted with a solvent consisting of 2% bovine serum albumin for 3 days at a flow rate of 30/hr, and the fraction No. 7 to 12 were collected, combined, and lyophilized to produce 8-(8-galactosidase)-dehydroxyseri by the reaction between the thiol group of 3-galactosidase and the aziridine nucleus of azilylglycine-insulin. Powder containing Rouglycil-insulin 252-9
(Max 27 sleeve plates) was obtained. '3l 8-(8-galactosidase)-dehydroxyseryl-glycyl-insulin in the precipitate obtained by the two-antibody method using
-About the galactosidase activity enhancing medium In the above method, a second guinea pig antibody is applied to the 8-galactosidase-binding immune component and the 8-(3-galactosidase)-dehydroxyseryl-glycyl-insulin obtained in step ① above. Using the precipitate obtained at least, mercaptoethane 2 skin M was added as a stabilizer, and 8-
A substance for enhancing 8-galactosidase activity was prepared using methanol, ethanol, and acetone as galactosidase activity enhancing media.

Medium and amount used: Mercabbutethane, without active additives
Added 100 methanol 10 scales
Added 253 Methanol 15 more Added 250 Methanol 20* Added 212
Ethanol 10* Addition 185 ethanol 1
5* Added 192 ethanol 20 taku
Addition 150 Acetone 10* Addition 148
Acetone 15% addition 152 Acetone 20* addition 125 As a result, in the B-galactosidase activity enhancement medium, the amount of these used is approximately 10-15% of the total amount.
It is good to use the degree.

A precipitate obtained by reacting 3-(8-galactosidase)-dehydroxyserylglycyl-insulin with a second guinea pig antibody was obtained as follows.

B-(8-galactosidase)-dehydroxyseryl-glycyl-insulin obtained from (21) above 0.4
mU. / 100 feet of seawater, serum obtained from guinea pigs (
2500p dilution: 1st antibody) 100p, gelatin
Veronal buffer (pH 7.4) 200ml, total 500ml
Let the Buddha stand overnight, then add normal guinea pig serum (10
Add 100 ml of “sound dilution” and further add 1 g of guinea pig
Eel antiserum sensitized with (4-fold dilution: second antibody) 10
300 ratio pm after adding 0 overnight and leaving it overnight, 1
The precipitate was collected, washed with phosphate buffer (pH 7.4), and used for this test.

As shown in the various examples above, B of the present invention
- The hydrophilic alcohols and ketones used as galactosidase activity enhancing media should be 5 to 15% of the total amount.
%, and it is particularly preferred to use methanol, furthermore in conjunction with a compound having a thiol group such as mercaptoethane, furthermore in this activity enhancement, various 8-galactosidase binding monoimmune components can be used. The results showed good results. Example 2 [(lmM
Contains MgC12, 0.1M NaCl, 0.1% bovine serum albumin, 0.1% NaN3), 10% 9/secretion (buffer) o-nitrophenyl-B-D-galactoside 50ml, mercaptoethane 25hM, and methanol 0 .0
A 8-galactosidase activity enhancing medium for EIA was obtained by combining 25 and 25 ml of the same.

Example 3 o Nitrophenyl-8-D-galactoside 0.5 female,
Mercaptoethane 2 hM and methanol 0.025
8-galactosidase activity enhancement medium for EIA, which is prepared by combining 1mMgC12, 0.1MN
aC1, 0.1% bovine serum albumin, 0.1M NaN3
The 0.1 M phosphate buffer (pH 6.8) is adjusted and added to the above-mentioned 8-galactosidase activity enhancement medium as needed, preferably at a concentration of 0.25.

In addition, at the time of addition, 8-galactosidase binding-
It may be done at the time of adding the immune component, or it may be before or after. Example 4 Mercaptoethane 0.29 M, methanol 0.25 and lmM Shio 12, 0.1M NaCl, 0.1
% bovine serum albumin and 0.1M phosphate buffer (pH 6.8) containing 0.1% NaN3 were mixed to obtain a homogeneous 8-galactosidase activity enhancement medium for EIA.

In using this medium, it is usually sufficient to add 5 to 10 o of the substrate for the B-galactosidase-binding-immune component. Example 5lmMM poly12, 0.1M NaCl, 0.
1% bovine serum albumin, 250% of 0.1M phosphate buffer (pH 6.8) containing 0.1NaN3, 50% of o-nitrophenyl-8-D-galactoside, 29M of mercaptoethane and 25M of methanol were mixed to form a homogeneous solution. None, this is used in the required amount as an enhancement medium for 8-galactosidase activity for EIA.

Example 6 The same procedure was carried out using glutathione, dithiothreitol, benicillamine, and cysteine in place of mercaptoethane in Example 5, respectively, to obtain an enhancing medium for B-galactosidase activity for EIA. It showed good results.

Example 7 The same procedure was carried out using acetone, ethanol, propylene glycol, and glycerin instead of methanol in Example 5 to obtain an enhancing medium for 8-galactosidase activity for EIA, and all of them gave good results. showed that.

Claims (1)

[Scope of Claims] 1. One or two types selected from the group consisting of hydrophilic alcohols and ketones as an immune reaction measurement system medium in enzyme immunoassay using β-galactosidase-binding immune component. A medium for enhancing β-galactosidase activity of a β-galactosidase-binding immune component in an enzyme immunoassay using a system containing 10 to 15% of the above compound based on the total amount and a compound having a thiol group. 2. The β-galactosidase activity enhancing medium according to claim 1, wherein the hydrophilic alcohol is an alcohol having 1 to 3 carbon atoms. 3. The β-galactosidase activity enhancing medium according to claim 1 or 2, wherein the hydrophilic alcohol is a mono- to trihydric alcohol. 4 Hydrophilic alcohols include methanol, ethanol,
Claim 1, or 2 or 3, which is propanol, propylene glycol or glycerin.
Enhancement medium for β-galactosidase activity as described in . 5. The β-galactosidase activity enhancing medium according to claim 1, wherein the hydrophilic ketone is acetone or methyl ethyl ketone. 6. The β-galactosidase activity enhancing medium according to any one of claims 1 to 5, which contains a compound having a thiol group and/or a buffer. 7. The β-galactosidase activity enhancing medium according to claim 6, wherein the compound having a thiol group is glutathione, methylcaptoethane or dithiothreitol. 8. Claims 1 and 2 comprising a system containing methanol or ethanol in an amount of 10 to 15% based on the total amount, 10 to 25 mM of glutathione, ethyl mercaptan or dithiothreitol, and a buffer solution. , 3rd
The β-galactosidase activity enhancing medium according to item 1, 4, 6, 7 or 8.