GB2089980A - Method for assaying superoxide dismutase - Google Patents

Abstract

In a method for assaying superoxide dismutase (SOD), a specimen containing SOD is reacted with an SOD-enzyme conjugate and an antibody against SOD, and the SOD level of the specimen is determined by measuring the enzyme activity in either the solid or liquid phase of the reaction mixture. The enzyme may be beta-galactosidase.

Description

SPECIFICATION Method for assaying superoxide dismutase The present invention relates to the method for assaying superoxide dismutase (hereinafter abbreviated as SOD). More particularly, it relates to the method of the enzyme immunoassay for SOD using a competition technique.

SOD is an enzyme which catalyzes the dismutation or disproportionation of superoxide free radical anions (0,:) and its assay has come into the limelight from the clinical standpoint in recent years. As to arteriosclerosis, particularly, its relationship to lipid superoxide has come to be clarified. It is said that lipid superoxide increases with the advance in age.

Under such circumstances, the microanalysis of SOD in tissues, such as liver and lung, has been highly desired. Also, SOD comes to be used as various therapeutic agents, so that the fractional quantitation of SOD in various animals and the measurement of SOD level in the tissues have been desired.

Concerning the conventional SOD assay, the quantitative method of McCord et al [J. Biol. Chem., 224, 6049-6055 (1 969)j has been proposed, which, however, is troublesome in its procedure and insufficient in its measurement sensitivity.

As the results of exhaustive investigations on the simple and excellent method for assaying SOD, the present inventors have found that there is least cross-sensitivity among various SOD derived from different species of animals as to the specific conjugation of the SOD with an acceptor which is specifically conjugative to SOD, and they have accomplished a satisfactory method for assaying SOD by utilizing an SOD-enzyme conjugate obtained by the conjugation of SOD with a labelling enzyme.

In the accompanying drawings, Fig. 1 shows a curve showing antibody titre of antiserum against orgotein, Fig. 2, a curve showing antibody titre of antiserum against human SOD, Fig. 3, a calibration curve of orgotein, and Fig. 4, a calibration curve of human SOD.

First of all, SOD employed in the present invention may be any of those having the activity to catalyze the dismutation or disproportionation of the superoxide free radical anions. For example, SOD derived from bovine, particularly, orgotein which is an SOD derived from bovine liver, SOD derived from human, and other SOD from various animals, may be illustrated. They may be suitably separated and purified according to the conventional method. Thereafter, the resulting SOD is conjugated with a labelling enzyme. As the enzyme, there may be suitably employed oxydoreductase, hydrolase, invertase, lyase, isomerase and ligase.There may be exemplified lactate dehydrogenase, maleate dehydrogenase, malate dehydrogenase, maltose dehydrogenase, lactate oxidase, maleate oxidase, peroxidase, glucose oxidase, choline oxidase, xanthine oxidase, amino acid oxidase, sarcosine oxidase, catalase, a-amylase, i3-galactosidase, lysozyme, lipase, alkali phosphatase, aminopeptidase, trypsin, papain, a-chymotrypsin, amidase, hexokinase, glycerokinase, etc. Further, any spacer may be introduced in advance to these enzymes.The enzymes may be modified by introduction of aldehyde, amino or thiol group by use of a spacer-introducing agent, for example, dialdehyde, such as glutaraldehyde, a reactive derivative, such as amino acid chloride, dialdehyde or dicarboxylic acid chloride plus or diamine, such as hexamethylenediamine or decamethylenediamine, S-acetylmercaptosuccinic anhydride, dialdehyde plus 2-aminoethanethiol and the like. Similarly, SOD may also be modified in advance by introducing an optional spacer.

In preparing the SOD-enzyme conjugate through conjugation of SOD and a labelling enzyme, the conjugation may be effected through amino, hydroxyl, thiol or carboxyl group in SOD or the labelling enzyme, or aldehyde, amino, thiol or carboxyl group which has been newly introduced in SOD or the enzyme. The conjugation of the both components may be direct, or indirect using a conjugating agent.

For example, carboxyl or amino group in a labelling enzyme may be reacted with amino or carboxyl group in SOD in an inert medium in the presence of a condensing agent such as an aqueous soluble carbodiimide. Or, the reaction may be effected based upon the reactivity between aldehyde and amino groups.In preparing SOD-enzyme conjugate using a condensing agent, the latter may be any of broadly known multifunctional compounds, for example, diisocyanate compounds, such as hexamethylene diisocyanate and 2,4-toluene diisocyanate; diisothiocyanate compounds such as hexamethylene diisothiocyanate; dialdehydes such as succinaldehyde, glutaraldehyde and adipaldehyde; N,N'ethylenebismaleimide; N,N'-o-phenylenedimaleimide; bisdiazobenzidine; N,N'polymethylenebisiodoaceta mide; diethyl malonimidate; dimethyl adipinimidate; sulfidocarboxylic acids such as 3-(2'-benzothiazolyl-dithio)propionic acid, 3-(2'-pyridyl-N-oxido-dithio)propionic acid and 6 N[3-(2'-benzothiazolyldithio)propionyl]caproic acid, and their reactive derivatives such as succinimide esters, p-nitrophenyl esters, acid chlorides and imidates (see Japanese Patent Application No.

85900/1978); and maleimidocarboxylic acids such as maleimidobenzoic acid, maleimidophenylacetic acid and maleimidophenylpropionic acid, and their reactive derivatives.

To obtain the conjugate, the reaction may ordinarily be carried out in a buffer solution of pH 6 to 8 in the presence of an organic solvent such as methanol, ethanol, acetone, dioxane, dimethylsulfoxide, dimethylacetamide, tetrahydrofuran and the like, or in such an organic solvent, at a temperature of 0 to 400 C. After the reaction, the conjugate may be purified by an adsorption chromatography or gel filtration procedure, as required.

As the acceptor specifically conjugative to SOD, an antibody obtained when SOD as antigen is sensitized to another kind of animal, or an antiserum containing such antibody, may generally be used.

For example, an emulsion of orgotein derived from bovine in Freund's complete adjuvant may be injected to a different kind of animal, such as rabbit and guinea pig, several times in a definite period of time, to give an immuno-sensitization. Thereafter, the blood is collected and treated according to the conventional method such as centrifugation, to obtain an antiserum. Further, the antibody may be separated from the antiserum according to the conventional method such as salting-out, isoelectric point precipitation, dialysis, chromatography, gel-filtration and others.

The resulting antibody may be employed on an insoluble carrier, if required, for example, insoluble protein carrier such as albumin and gelatin; insoluble semisynthetic high molecular carrier such as agarose, cellulose and dextran, treated with epichlorohydrin or bromocyan, or those further treated with an amination agent; and insoluble synthetic high molecular carrier such as polymers or copolymers of acrylonitril, acrylic acid, acrylate esters, methacrylic acid, methyl methacrylate, vinyl alcohoi, vinyl acetate, aminostyrene, acrylamide, ethylene, maleic anhydride, and the like. The antibody may be bound with these carriers through a multifunctional compound as described above.

In carrying out the method of the present invention by use of a SOD-enzyme conjugate, a solution containing a definite amount of the SOD-enzyme conjugate, a solution containing an acceptor specifically conjugative to SOD, and a specimen of which the SOD value is to be assayed, are mixed together, and allowed to react overnight generally at temperature of 40C. In this reaction, an aqueous medium, preferably, a buffer solution, such as 0.01 M phosphate buffer solution (pH 7.2) containing 0.25% of bovine serum albumin (BSA), O.-1 % of NaN3 and 0.15 M of NaCI, may be employed. The aqueous medium may also be used as a dilution medium for each agent mentioned above. After the reaction, the solid and liquid phases are separated from each other.In a preferred example, an immune globulin from the animal species from which the acceptor specifically conjugative to SOD is generated, and an antibody against the globulin of the animal species or an antiserum containing it, may be added thereto, and the mixture is allowed to react overnight at a temperature of 40C. After the reaction, the solid and the liquid phases are separated from each other by a separating procedure such as centrifugation. Then, the enzyme activity in the solid phase (B component) or in the liquid phase (F component) may be assayed.

In assaying the enzyme activity, the exhausted constituent or the formed constituent may be measured, using a substrate corresponding to the enzyme reaction employed. In case of an oxidase, for example, SOD may be assayed by measuring the exhausted enzyme or the formed hydrogen peroxide after the substrate is oxydized by the enzyme reaction. From this enzyme activity value, SOD in the B component or the unreacted SOD-enzyme in the F component is quantitated to determine the amount of the SOD-enzyme conjugate exhausted after the reaction. In case of a reductase, it is preferred to measure the coenzyme exhausted after the enzyme reaction. In case of a hydrolase, the hydrolyzate formed after the enzyme reaction may be measured.For these measurements, various known methods can be used, for example, the direct measurements using oxygen electrode, hydrogen peroxide electrode and colorimetry, or the indirect measurement utilizing the color change after the reaction by use of hydrogen peroxide-coloring agent, and such methods may suitably be selected according to the kind of enzyme employed.

By practicing the present invention as mentioned above, SOD in a specimen can be assayed with full satisfaction. Thus, the present invention is the favorable method for assaying SOD, with the safety and stability of the various agents employed.

The present invention will be described more concretely hereinafter with respect to the following example which, however, should not be construed to be limitative.

EXAMPLE (1) SOD 1) SOD derived from bovine (orgotein): procured from Diagnostic Data Incorporated.

2) SOD derived from human (human SOD): separated from human erythrocyte and purified, according to the method described in J. Boil. Chem., 234, 46 (1959).

3) SOD derived from other animals: separated and purified according to the similar method as in the separation and purification of human SOD.

(2) Acceptor specifically conjugative to SOD 1) Orgotein (5 mg./ml.) was blended and mixed with the equal volume of Freund's complete adjuvant to prepare an emulsion. Each 0.5 ml. aliquot of the emulsion was injected subcutaneously to rabbits at the back, 3 times biweekly and, with a 3 week interval, 3- times biweekly, to form the immunological sensitization. Then, the whole blood was collected when its antibody titre reached 800 U during the course of assay according to the complement fixation, and the antiserum against orgotein was obtained according to the conventional method.

2) Human SOD (5 mg./ml.) was immunized to rabbits in the similar way as in orgotein in the item 1). The blood was collected and the antiserum against human SOD was obtained.

3) The cross reactivity between each resulting antiserum and SOD derived from the different kinds of animals were investigated. The results are as illustrated in Table 1, which shows that the cross reactivity between the resulting antisera and SOD derived from the different kinds of animals are below 1/1000.

TABLE 1

Antiserum Antisentm against against argotein human SOD 1 Orgotei n 1 below I'iO Human SOD below 1 1 1000 Swine SOD below 1 below 1 1000 1000 Rabbit SOD below 1 below 1 5000 1000 Rat SOD below 1 below 1 s000 1000 Equine SOD below 1000 below 1000 (3) SOD-enzyme conjugate 1) A mixture of 50 mM phosphatabuffer solution (pH 8.0) containing 30 mg. of orgotein and 1 mM of ethylenediaminetetraacetic acid (EDTA), with 360 yl. of dimethylformamide containing 1 0O ug. of succinimide ester of 3-(2'-benzothiazolyl-dithio)propionic acid was allowed to react by 1 hour stirring under ice-cooling. Then, to 2 ml. of 100 mM phosphate buffer solution (pH 7.0) was added 20 l. aliquot of the reaction mixture, and then 5 mg. of ,B-galactosidase was added. The mixture was allowed to react by 30 hour stirring under ice-cooling.After the reaction, the mixture was charged into a Cephadex G-1 50 column (2 x 70 cm.), eluted and gel-filtered with 100 mM phosphate buffer solution (pH 7.0) to recover a 68 to 77 ml. fraction having ,B-galactosidase activity. Orgotein-P-galactosidase conjugate was obtained by freeze-drying of the fraction (96% of p-gaíactosidase activity yield).

The ,B-galactosidase activity was determined from the extinction at 420 nm, using o-nitrophenyl ,B-galactosidase as the substrate.

2) According to the method described in J. Biochem., 79, 233-236 (1976), to a solution of 30 mg. of human SOD in 1 ml. of 50 mM phosphate buffer solution (pH 7.0) was added 1 ml. of tetrahydrofuran solution containing 5 mg. of succinimide ester of methamaleimidobenzoic acid and the mixture was allowed to react for 30 minutes at the room temperature. The mixture was charged into a Cephadex G-25 column (1 x 70 cm.) and gel-filtered using 50 mM phosphate buffer solution (pH 7.0) as the solvent, to recover a 1 6-22 ml. fraction (containing 22 mg. of SOD). The fraction was freezedried, and 0.7 mg. aliquot of the dried substance was dissolved in 0.2 ml. of 50 mM phosphate buffer solution (pH 7.0).To the resulting solution was added 0.3 ml. of 50 mM phosphate buffer solution (pH 7.0) and then with 0.5 mg. of ,3-galactosidase. The mixture was allowed to react for 2 hours at the room temperature. After the reaction, the mixture was gel-filtered and freeze-dried to obtain human SOD-p- galactosidase conjugate (78% of/3-galactosidase activity yield).

(4) Anti-rabbit- > s-globulin antibody Rabbit y-globulin (10 mg./ml.) was blended and mixed with the equal volume of Freund's complete adjuvant, to prepare an emulsion. Each 0.5 ml. aliquot of the emulsion was injected subcutaneously to goats at 4 places of the back. After biweekly 6 times immunization, followed by 2 week interval, the whole blood was collected, from which anti-rabbit-y-globulin goat serum was obtained according to the conventional method.

(5) Assay of antibody titres of antisera against orgotein and against human SOD 1 ) Assay of antiserum against orgotein A mixture of 100 l. of orgotein-P-gaiactosidase conjugate-containing solution, 100 yí. of a diluted solution of antiserum against orgotein and 100 ,zl. of orgotein-containing solution was allowed to react overnight at temperature of 40C and thereafter thereto was added 100 ,ul. of 50 g./ml. rabbit IgG-containing solution and 100 yI. of anti-rabbit-y-globulin goat serum (8 time dilution), and the mixture was allowed to react overnight at temperature of 4"C. [All soivents employed were 10 mM phosphate buffer solution (pH 7.0) containing 0.25% of bovine serum albumin, 0.1% of NaN3 and 0.15 M of NaCI.] After the reaction, to the mixture was added 4 ml. of physiological saline and subjected to centrifugation at 3000 r.p.m. for 1 5 minutes, to recover the solid substance as the precipitate. To the solid substance was added 0.2 ml. of 0.03 M phosphate buffer solution (pH 7.0) containing 4 mg./ml. of o-nitro-phenyl-B-galactoside, 20 mM of mercaptoethanol and 10% of methanol. The mixture was allowed to react for 1 hour at temperature of 370C, and then to the mixture was added 2.3 ml. of glycine-NaOH buffer solution (pH 1 1.5). The color change was measured from the extinction at 420 nm wave length.

As the results, antibody titre of antiserum against orgotein was as shown in Figure 1, in which the curve represents the antibody titre of antiserum against orgotein.

2) Assay of antibody titre of antiserum against human SOD Using 100 ,ul. of human SOD-p-galactosidase conjugate-containing solution, 100 yl. of a distilled solution of antiserum against human SOD and 100 iul. of a human SOD-containing solution, with otherwise the same way as the assay of antiserum against orgotein as mentioned above, the antibody titre of the antiserum was assayed.

The results were as shown in Figure 2, in which the curve represents antibody titre of antiserum against human SOD.

(6) Assay of SOD 1) Assay of orgotein (calibration curve) In the similar way as in the assay of antibody titre of antiserum against orgotein, a mixture of 100 yí. of orgotein-P-galactosidase conjugate-containing solution (1.22 ngiml. of orgotein), 100 ,ul. of antiserum against orgotein (51200 time dilution) and 10owl. of a liquid specimen (containing 0 to 100 ng./ml. of orgotein), was allowed to react overnight at temperature of 40C. To the mixture was then added 100 yí. of a solution containing 50 yg./ml. rabbit IgG and 100 yI. of anti-rabbit-y-globulin goat serum (8 time dilution); and the mixture was allowed to react overnight at temperature of 40C.After the reaction, to the mixture was added 4 ml. of physilogical saline and subjected to centrifugation at 3000 r.p.m. for 1 5 minutes. To the recovered precipitate was added 0.2 ml. of 0.03 M phosphate buffer solution (pH 7.0) containing 4 mg./ml. of o-nitrophenyl-p-galactoside, 20 mM of mercaptoethanol and 10% of ethanol, and the mixture was allowed to react at temperature of 370C for 1 hour. After the reaction, to the mixture was added 2.3-ml. of glycine-NaOH buffer solution (pH 1 1.5), and the color change was measured from the extinction at 420 nm wave length As the results, an excellent calibration curve of orgotein was obtained, as shown in Figure 3.

2) Assay of human SOD (calibration curve) Using 100 yí. of human SOD-galactosidase conjugate-containing solution (1.14 ng./ml. of SOD derived from human), 100 ,ul. of antiserum against human SOD (640,000 time dilution) and a liquid specimen (containing O to 100 ngimí. of human SOD), with otherwise the similar way as in the assay of orgotein mentioned above, human SOD in the specimen was assayed.

As the results, an excellent calibration curve was obtained, as shown in Figure 4.

3) Assay of orgotein Orgotein was administered to equines at their knee-joint. (The doses are shown in Stable 2.) Using the serum from the equines, as the specimens, with otherwise the same way as the assay or orgotein (calibration) mentioned above, the level of orgotein in the equine serum after the orgotein administration was assay and calculated based upon the calibration curve.

The results were as shown in Table 2.

TABLE 2

Content of orgotein in serum (ng./ml.) Dose of Collecting time (hour) No. orgotein (mg.) 0.5 1 2 4 3 24 72 1 20 1,5 2.6 10.4 10.8 8.1 2.17 0.5 2 10 1.6 2.3 3.8 6.9 7.0 2.3 < 0.5 10 2.21 2.4 4.3 - 7.8 6.2 2.45 < 0.5 4 20 3.9 6.1 7.5 13.0 9.4 2.6 0.96 5 10 1.0 2.04 2.52 2.6 1.8 0.67 < 0.5 6 10 0 0 0 0 0 0 0 (detecting limit: 0.5 ng./ml.)

Claims (4)

1. A method for assaying superoxide dismutase comprising using a superoxide dismutase-enzyme conjugate obtained by the conjugation of superoxide dismutase and a labelling enzyme.

2. The method for assaying superoxide dismutase according to the Claim 1, wherein a superoxide dismutase-enzyme conjugate, a specimen and an acceptor specifically conjugative to superoxide dismutase, are allowed to react each other; the solid and liquid phases of the reaction mixture are separated from each other; and the enzyme activity in the solid or liquid phase is measured.

3. A method for assaying superoxide dismutase substantially as hereinbefore specifically described.

4. A superoxide dismutase material a portion of which has been assayed by a method according to either of claims 1 to 3.