FR2496693A1 - PROCESS FOR DETERMINING PEROXIDE DISMUTASE - Google Patents

Abstract

METHOD FOR DETERMINING PEROXIDE DISMUTASE. IT CONSISTS OF ADDING TO A SAMPLE A MATERIAL IN SOLID PHASE PREPARED FROM AN ACCEPTOR COMBINING SPECIFICALLY WITH PEROXIDE DISMUTASE, LET THE TWO CONSTITUENTS REACT ONE ON THE OTHER, ADDING A CONJUGATION PRODUCT TO THE REACTION PRODUCT OF AN ENZYME AND OF AN ACCEPTOR COMBINING SPECIFICALLY WITH PEROXIDE DISMUTASE, TO LET THEM REACT ONE ON THE OTHER AND TO DETERMINE THE ENZYMATIC ACTIVITY OF THE MATERIAL IN THE SOLID PHASE. MEDICINE.

Description

The present invention relates to a method of

  determination of peroxide dismutase (abbreviated hereinafter as POD).

  More particularly, the invention relates to a

  immunoassay by enzymatic POD using a sandwich technique. POD is an enzyme that catalyzes the disproportionation or dissociation of free-radical peroxide anions (2 '). In recent years, its dosage has become more and more

  attention from a clinical point of view and we would like

  The ability to perform microanalysis of POD in tissues such as the liver and lung. The POD has also come to be used as a therapeutic agent, so that one wishes to be able to perform the dosage of the rate

in the blood.

  With regard to the conventional dosage of POD,

  the method of McCord et al. [J.

  Biol. Chem. 244, 6049-6055 (1969)] which, however, is tedious in its operating protocol and insufficient

in its measurement sensitivity.

  The attached drawing shows the calibration curve

of orgotine.

  After much research into a simple and accurate method of determining POD, it has now been found that there is less cross-reactivity between the various PODs from different animal types for the POD-specific conjugate product. of an acceptor which conjugates specifically to the POD and the invention thus aims at a method of quantitative determination of the POD by adding

  a liquid sample to a prephase solid phase material

  from an acceptor, conjugating specifically to POD, to provoke a reaction, and then adding to the reaction product an acceptor-enzyme conjugate product

  to provoke a reaction and then to determine the activity

  enzymatic activity in the solid phase material.

  First, the POD used according to the invention may be any of those having the activity of catalyzing the disproportionation or dissociation of free radical peroxide anions. This is the case, for example,

  mention may be made of beef POD, in particular

  bind orgotine, which is a POD from beef liver, POD from humans and other POD from various animal species. The POD can be separated and purified from an animal correctly selected by the method described

  in J. Biol. Chem., 234, 46 (1959).

  To obtain the specific acceptor

  In POD, one can generally use an antibody obtained while the POD serving as an antigen is injected into another type of animal. This is the case, for example,

  orgotine emulsion from beef liver in the adjuvant

  Freund's complete sample can be injected at a different type.

  animal than rabbit and guinea pig, many times

  during a defined period to obtain immunosensitivity

  lisation. The blood is then collected and treated

  the conventional process, such as by centrifugation,

  droplet, isoelectric point precipitation, dialysis, chromatography, gel filtration, etc., to separate and purify the antibody. In addition, the acceptor thus obtained, which specifically conjugates to the POD, is attached to an insoluble support directly or indirectly using a fixing agent to obtain the solid phase material. The insoluble supports used to obtain the solid phase material may be those having a radical

  functional, for example, proteinaceous

  such as albumin and gelatin treated with glutaraldehyde, insoluble semi-synthetic

  high molecular weight, such as agarose, cellulose,

  and dextran treated with epichlorohydrin or

  cyanogen bromide, or those further treated with an amine reagent

  synthetic insoluble carriers of molecular mass

  such as polymers and copolymers of acrylonitrile, acrylic acid, acrylate,

  methacrylic acid, methyl methacrylate, vinyl alcohol

  vinyl acetate, styrene, aminostyrene, divinylbenzene, acrylamide, ethylene, maleic anhydride, crotonic acid, etc., and those further processed.

  by an amination reagent and insoluble mineral carriers

  such as silane compounds treated with a reac-

  tif of amination. Any of the various known methods can be used to introduce an amino group on the insoluble support. For example, amination by reduction of the nitro group, introduction of an aminoalkyl group by

  reaction of a hydroxy group on a γ-aminopropyltriethoxy

  silane at the introduction of the amino group by

  conversion of the hydroxymethyl group to the aldehyde group by periodate oxidation, followed by the reaction of the aldehyde with a diamine, such as hexamethylenediamine and decamethylenediamine, with the conversion of the hydroxyl group

  from a polysaccharide to an imidocarbonate group

  on cyanogen bramide, conversion of the amide group to an iminohalogenide group by a halogenated phosphorus compound. These functional groups can be reacted with an agent to introduce a new functional group, for example onto a dialdehyde, such as glutaraldehyde and adipaldehyde, on a reactive derivative such as a chloride

  of W-amino acid and on S-acetylmercapto acid anhydride

  succinic. To prepare the solid phase material, an acceptor specifically conjugating to POD can be attached to the insoluble support by the functional group of the molecule such as amino, aldehyde, carboxy, hydroxy, thiol, imidocarbonate, iminohalide, and the like. We can fix

  two-constituent to each other directly or indirectly-

  using a fixing agent. For fixing

  directly, the carboxy or imido group can be reacted

  carbonate of an insoluble support on the amino group of an acceptor molecule in an inert medium, if necessary in the presence of a water-soluble carbodiimide. For

  to obtain solid phase material by indirect fixation

  using a fixing agent, the latter can be any of the well-known multifunctional compounds,

  for example, diisocyanate compounds such as diisocyanate

  hexamethylene nate and toluene 2,4-diisocyanate; diisocyanates such as hexamethylene diisocyanate;

  dialdehydes such as succinaldehyde, glutaral-

  dehyde and adipaldehyde; N, N'-ethylenebismaleimide; N, N'-ophenylenedimaleimide; bisdiazobenzidine; N, N'polymethylenebisiodoacetamide; diethyl malonimidate; dimethyl adipinimidate; sulfuric acids

  such as 3- (2'-benzothiazolyl)

  dithio) propionic acid, 3- (2'-pyridyl-N-oxido-dithio) -

  propionic acid, 6-N [3- (2'-benzothiazolyl-dithio)

  pionyl] caproic acid and their reactive derivatives such as their succinimide esters, their p-nitrophenyl esters, their acid chorides and their imidates (see request

  Patent No. 85900/1978); and maleic acids

  such as maleimidobenzoic acid, acetic acid,

  maleimidophenylacetic acid and maleimidophenylpropionic acid

and their reactive derivatives.

  As the enzyme used to obtain the conjugate product

  With an acceptor conjugating specifically to POD, an oxidoreductase, a hydrolase, an invertase, a lyase, an isomerase and a ligase can be used. By way of example, mention may be made of lactase dehydrogenase, maleate dehydrogenase, malate dehydrogenase, maltose dehydrogenase, peroxidase, lactate oxidase, maleate oxidase, glucose oxidase, choline oxidase, xanthine oxidase, amino acid oxidase, sarcosine oxidase, catalase, α-amylase, e-galactosidase, lysozyme, lipase, alkali phosphatase, aminopeptidase, trypsin, papain, α-chymotrypsin, amidase,

  hexokinase, glycerokinase, etc. In addition, we can introduce

  in these enzymes, to leave a congestion agent in advance.

  The enzyme can be modified by introducing a radical

  aldehyde, amino or thiol using an encapsulation agent

  for example a dialdehyde such as glutaraldehyde, a reactive derivative such as a w-amino acid chloride, a dialdehyde or a dicarboxylic acid chloride added

  diamine such as hexamethylenediamine and

  methylenediamine, S-acetylmercapto acid anhydride

  succinic, dialdehyde supplemented with 2-aminoethanethiol, etc. Similarly, the acceptor can be modified in advance by

  introducing a congestion agent.

  To prepare the acceptor-enzyme conjugate product, the two components can be conjugated via an amino, hydroxy, thiol or carboxy group of the acceptor or enzyme, or via an aldehyde group. , amino, thiol or carboxy that has been produced in the acceptor

  or in the enzyme. We can also combine the two

  killing using the multifunctional fixation agent

as mentioned above.

  To obtain the solid phase material, or the

  of the enzyme-acceptor conjugate, it can usually be

  the reaction in a buffer solution of pH 6 to 8 in the presence of an organic solvent such as methanol, ethanol, acetone, dioxane, tetrahydrofuran, dimethylacetamide, dimethylsulfoxide, etc., or in such an organic solvent at a temperature of 0 to 400C. Once the reaction is complete, the product can be washed and purified if necessary. Purification is preferably carried out

  using adsorption chromatography or a filter

gel.

  In carrying out the process according to the invention, a defined amount of the solid phase material prepared from an acceptor conjugating specifically to POD and a sample must measure the value of the POD. The first reaction can generally be carried out at a temperature of 40C and in an aqueous medium, for example in a 10mM phosphate buffer solution (pH 7.2) containing 0.25% serum albumin of beef (BSA). 0.1% NaN3 and 0.15 M NaCl. Once the reaction is completed, the solid phase material can preferably be washed and then added to it in a certain amount.

  of a conjugated product of an enzyme and an acceptor

  judging specifically at the POD. The mixture is left

  run in an aqueous medium, usually at a temperature of 40C, overnight. Then we collect the material in

  solid phase, washed and assayed for enzymatic activity.

  tick. By assaying the enzymatic activity, it is possible to measure the constituent consumed or the constituent formed using

  a substrate corresponding to the enzymatic reaction employed

  Yee. In the case of an oxidase for example, the POD can be assayed in the sample by measuring the enzyme consumed or the hydrogen peroxide formed after the substrate has been oxidized by the enzymatic reaction and determining the enzyme activity.

  from this value. In the case of a reduction

  it is better to measure the coenzyme consumed after the enzymatic reaction. In the case of a hydrolase,

  the hydrolyzate formed after the enzymatic reaction can be measured

  matic. For these measurements, various methods can be used

  known, for example, direct measurements using electricity

  oxygen scavenger, the hydrogen peroxide electrode and the color

  metric, or the indirect measure using the change of

  staining after the reaction on the hydrogen peroxide mixture

  coloring agent and these procedures can be

  according to the nature of the enzyme used.

  By practicing as mentioned above, one can assay the POD of a sample in a quite satisfactory manner. This is why the invention is a process

  very favorable dosage of POD, the various agents used

are safe and stable.

  The following example illustrates the invention.

EXAMPLE

(1) POD.

  1) POD from beef (orgotine): supplied by

  Diagnostic Data Incorporated.

  2) POD from human (human POD): separated from human erythrocyte and purified by the method described in

J. Biol. Chem., 234, 46 (1959).

  3) POD from other animals: separated and purified by a process similar to separation and

the purification of the human POD.

  (2) Acceptor specifically conjugating to the POD.

  1) Orgotine (5 mg / me) is mixed with one volume

  equal Freund's complete adjuvant to prepare an emulsion.

  Each 0.5-ml aliquot of the emulsion is injected subcutaneously into rabbits, in the back, three times every two weeks and after an interval of three weeks.

  three times every two weeks to get the feeling

  immunological recognition. All the blood is then collected when its antibody titre reaches 800 U during the course of the assay, following the complementary fixation, and the antiserum is obtained against the orgotin according to

classical process.

  2) Human POD (5 mg / mt) is injected into rabbits in the same way as orgotine in paragraph 1

  above. We collect the blood and we get the anti-

serum against human POD.

  3) the cross-reactivities between each antiserum obtained and the POD obtained from different types of animals are sought. The results obtained are given in Table 1

  which shows that the reactivities crossed between the anti-

  sera obtained and POD from various types of animals

ailments are less than 1/1000.

TABLE 1

  (3) Solid phase material prepared from the acceptor

  conjugating specifically to orgotine.

  Benzene was washed with seventy-five pieces (100 g) of Nylon-6,6 grains (9 mm cylindrical form of antiserum Antiserum against orguein POD human Orgotine 1 lower 1OOO lower human POD 1OO0 1 POD lower pig 1 lower Lower rabbit POD 500o lower Tooo

5000 1000

  POD lower rat lower 1 dchvi5000i 1000 POD lower horse 1 lower

1000 1000

  diameter and 5 mm in thickness) and 200 gram

  of benzene containing 18 grams of phosphorus pentachloride, and the grains are allowed to react for two days with stirring at room temperature and then washed well with benzene. The grains are further washed with a small amount of tetrahydrofuran, and then 1 ml of tetrahydrofuran containing 29 grams of adipic acid is added.

  The mixture is left to react for one night at

  room temperature. The grains are then washed thoroughly

  dimethylformamide and place them in 100 niml of a solution

  dimethylformamide containing 2.3 grams of N-hydrochloride

  xysuccinimide and 4 grams of N, N'-dicyclohexylcarbodiimide.

  The mixture is allowed to react for 6 hours at room temperature.

  ambient temperature. The grains are washed with dimethylformamide and 100 ml of an aqueous solution of 200 mg hexamethylenediamine (pH 11.0) are added. The seeds are allowed to react overnight at room temperature, then washed with 0.5 M saline and again with saline, and reacted with additional salt.

  a solution containing glutaraldehyde, and then washed.

  On the other hand, a rabbit γ-globulin fraction containing the antibody against orgotine is obtained from the antiserum against the above-mentioned orgotin by ammonium sulfate fractionation, according to the conventional method. . The fraction is added to the grains in an amount corresponding to 100 μg per piece of grain and allowed to react. (The amount of γ-globulin fixed is 65 μg per

average room).

  (4) Conjugate product of an enzyme and an acceptor

  conjugating specifically to orgotine.

  Using rabbit y-globulin fraction

  containing the antibody against orgotin, obtainoetel that men-

  tioned above, and 0-galactosidase, we obtain a

  solution of the conjugate product of the antibody against the organism

  protein and e-galactosidase according to the process described

  in J. Biochem., 84, 93-102 (1978) (using N, N'-

  o-phenylenedimaleimide as multifunctional agent).

  (5) Dosage of orgotine (calibration curve).

  To a part of the grains, obtained as mentioned above, (the solid phase material of the antibody against orgotine), 100 μl of a liquid sample (containing from 0 to 100 mg / ml of orgotine) and 100 μl of a 10 mM phosphate buffer solution (pH 7.2) containing 0.25% serum albumin from beef, 0.1% NaN 3 and 0.15 M NaCe and left blank. The reaction is complete, the workpiece is washed with the same buffer solution, 100 μl of a solution containing a conjugate product of the antibody against orgotine are added to it. e-galactosidase, prepared as mentioned above (diluted 50-fold), and allowed to

  room react overnight at a temperature of 4 C.

  When the reaction is complete, the part is washed with physiological serum

  0.2 ml of a phosphate buffer solution is added to it.

  30 mM phate (pH 7.0) containing 4 mg / me of o-nitrophenyl-B-

  galactoside, 20 mM mercaptoethanol and 10% ethanol and allowed to react for 1 hour at a temperature of 37 C. 2.3 ml of a glycine-NaOH buffer solution (pH 11 , 5) and we measure the change of

  staining by extinction at the wavelength of 420 nm.

  An excellent calibration curve is thus obtained.

  age for orgotine, as shown in the drawing.

(6) Dosage of orgotine.

  By using, as samples, sera obtained from rats, after subcutaneous injection with each of them of 1 mg of orgotine, the same method of dosing of orgotine is repeated (for the calibration curve) to dose

  the level of orgotine in the serum of the rats, after

  treatment of orgotine, followed by a calculation

based on the calibration curve.

  The results obtained are given in Table 2.

TABLE 2

  Orgotin levels in sera (pg / mZ) No. Time (in hours) after which blood is collected

0.25 0.5 1 2 3 5 7

  1 0.72 0.91 0.85 1.22 1.51 0.66 0.65

  2 2.94 1.33 1.35 1.70 2.30 2.29 2.22

  3 0.90 1.20 3.52 2.32 2.30 2.23 1.05

  4 0.75 0.80 1.14 3.52 2.50 2.25 2.20

0.76 0.90 0.92 1.95 0.76 0.72 0.64

Claims (1)

CLAIM   Method for the determination of peroxide dismutase,   It consists in adding to a sample a solid-phase material prepared from an acceptor conjugating specifically to peroxide dismutase,   the two constituents react one on the other, to add   to the reaction product a conjugated product of an enzyme   and an acceptor specifically conjugating to the   oxide dismutase, allowing them to react with one another and determining the enzymatic activity of the solid phase material.