PROCEDURE FOR THE DETERMINATION OF PEROXIDASE ACTIVITY BY END DILUTION TITRATION AND MEANS FOR ITS REALIZATION

Abstract: An improved determination of peroxidase activity by end dilution titration is carried out with a benzidine deriva­ tive of the general formula I where R1 stands for a hydrogen atom, a halogen atom, an amino group, a (C₁-C₄) alkyl group, a (C₁-C₄) alkoxy group, a carboxy group, a sulfo group or a carboxypropyloxy group and where R2 stands for a hydrogen atom, a halogen atom, a (C₁-C₄) alkyl group, or a salt of this benzidine compound, in the presence of a non-chromogenic or low-chromogenic phenylene derivative of the general formula II where R3 and R4 are equal or different and stand for a hydroxy group or an amino group, a (C₁-C₄) alkyl amino group or a benzylamino group.

Description: The invention concerns a procedure for the determination of peroxidase activity and a reagent composition for its determination. In EP-A 0029917 a qualitative method for the detection of small amounts of peroxidase is described. The method is based on the known color appearance connected with the reaction of hydrogen peroxide with a benzidine type indicator in the presence of peroxidase. According to EP-A 0029917 the sensitivity of the test can be enhanced by the addition of an amine oxide. Peroxidase, e.g. horse radish peroxidase (HRPO) finds broad application as a marker enzyme in enzyme-immunological determination methods ( E nzyme i mmuno a ssay (EIA)). A common feature of all EIA systems is the exploitation of an antigen-antibody-interaction for the determination of the concentration of one of the reactants (antigen or antibody). A multitude of EIA systems have been described. These may be classified in systems with enzyme labelled antigen and in systems with enzyme labelled antibody. One example of an EIA system with labelled antigen is the competitive solid phase EIA ( E nzyme- l inked I mmuno S orbent A ssay (ELISA)). In this a known amount of enzyme-labelled antigen and the antigen to be determined (analyte) compete for the limited number of binding sites of a specific antibody which is immobilized on a solid phase. The higher the concentration of analyte, the lower the amount of enzyme-labelled antigen bound to the immobilized antibody. Making use of a calibration curve, the determination of the enzyme activity bound to the the solid phase by interaction with the antibody allows the determination of the analyte concentration. An exponent of the antibody-labelled EIA is the "Sandwich"-assay (or two-site enzyme immunometric test). The test includes two immunological reactions: In the first reaction a macromolecular antigen is bound more or less completely to the antibody immobilized on a solid phase. After removal of the reaction solution, the bound antigen is reacted with an excess of a conjugate consisting of a specific antibody and enzyme. The extent of the binding of enzyme-labelled antibody is proportional to the amount of antigen, thus the determination of the remaining enzyme activity after removal of excess conjugate is a measure of the amount of antigen. Thus, the determination of enzyme activity is an essential step in all EIA's. In general, enzyme activity is determined by well known procedures, typically by following the changes of the optical properties (absorption, fluorescence, luminescence) of an appropriate substrate effected by enzymatic action. Appropriate measuring instruments are used, and a linear proportionality between measuring signal and amount of enzyme is advantageous. For the determination of peroxidases, benzidine and its derivatives as o-dianisidine, o-tolidine, dicarboxidine and 3,3',5,5'-tetramethylbenzidine in combination with hydrogen peroxide are useful. The reference Z.med.Labor.-Diagn. 24 155-160 (1983) relates to the use of o-dianisidine or o-phenylenediamine as hydrogen donors in an automatized kinetic method for determination of horse radish peroxidase. For a variety of applications of EIA, e.g. in serological determinations of antibody-titers, the high precision achievable by instrumental measurement is only of little value, since only rises in concentration by a factor of 2 to 4 relative to a reference are considered meaningful, whereas the whole concentration range to be covered includes several orders of magnitude. For such determinations the method of end dilution titration is applicable. It consists in the determination of the highest dilution of the original solution which gives a positive result for the analyte of interest in a defined detection procedure. The reciprocal of this highest dilution (e.g. 1:1,000) is usually denoted as "titer" (e.g. 1,000). The numerical value is of course influenced by the sensitivity of the detection method in use which may be different from case to case due to fluctuations in the performance. This can be overcome by standardization of the titers against a reference of known or defined concentration which is conducted in every determination. In practice the sample solution is diluted serially with an appropriate diluent, normally in geometrical series (e.g. titer steps 1:10; 100; 1,000 ... or 1:2; 4; 8; ... or 1:1.5; 2; 3; 4; 6...). A variety of simple technical means are available for this purpose, e.g. the microtiter system. An equal volume of each titer step is subjected to the test reaction which in form of a yes-no answer leads to the identification of the highest titer. If a visual observable detection reaction is applied, no measuring instrument is needed. The applicability of a detection reaction is highly dependent upon the unambiguity with which a (weakly) positive result may be discerned from a negative result (yes-no-answer). But the detection of enzyme activities with chromogenic substrates usually leads to continuous increase (decrease) in color development with rising (falling) enzyme concentration, thus making almost impossible the clear-cut identification of dilution end points. The objective of the present invention is to provide a detection reaction for peroxidase which unambiguously indicates the surpassing (subpassing) of a certain amount of enzyme by an abrupt change in a visual observable property (color jump) and thus allows the unambiguous detection of end point titrations, i.e the clear-cut demonstration of the sub- or surpassing of a threshold concentration, and to provide a composition for the performance of this reaction. The object is fulfilled by providing an improved method for the determination of peroxidase activity by end dilution titration under peroxidase end point dilution titration determining conditions with a benzidine derivative in the presence of a color retarding amount of non-chromogenic or low-chromogenic proton donor to retard color development for a sufficient period of time to permit a sharp dilution endpoint and thereafter detecting peroxidase activity. Peroxidases of different origins may be determined, e.g. Japanese radish peroxidase, especially horse radish peroxidase (HRPO) (EC1,11.1.7.). Benzidine derivatives are those of the general formula (I) where R1 stands for a hydrogen atom, a halogen atom, an amino group, a (C₁-C₄) alkyl group, a (C₁-C₄) alkoxy group, a carboxy group, a sulfo group or a carboxypropyloxy group and where R2 stands for a hydrogen atom, a halogen atom, a (C₁-C₄) alkyl group, or the salts thereof. (C₁-C₄) alkyl groups are branched or unbranched alkyl groups, likewise the (C₁-C₄) alkoxy groups maybe branched or unbranched. The methyl and methoxy group are preferred. Halogen atoms include bromine, chlorine and fluorine; chlorine is preferred. Compounds of formula (I), where R1 stands for a hydrogen atom, a methyl group, or a methoxy group and where R2 stands for a hydrogen atom or a methyl group, are preferred. Especially preferred is the compound of formula (I), where R1 and R2 stand for a methyl group in the ortho position to the amino groups. Non-chromogenic or low-chronogenic proton donors are phenylene derivatives of the general formula (II) where R3 and R4 are equal or different and stand for a hydroxy group, an amino group, a (C₁-C₄) alkyl amino group or a benzylamino group. Preferred examples of the phenylene derivatives are hydroquinone, 1,2-dihydroxybenzene, resorcinol, o-, m-and p-phenylene diamine, 4-aminophenol, 4-methylaminophenol, 4-benzyl amino phenol. Selected phenylene derivatives (II) are o-phenylendiamine and 4-aminophenol, especially hydroquinone. As mentioned, procedures for determining peroxidase activity by end dilution titration are well known and are carried out under peroxidase end dilution titration determining conditions. Likewise, the improved procedure of this invention is carried out under peroxidase activity end dilution titration determining conditions utilizing a sufficient amount of the benzidine derivative, usually an excess amount of hydrogen peroxide or its adducts. The hydrogen peroxide can be added to the reaction or generated in situ. Such conditions include suitable reaction temperatures, e.g. 4 °C to 37 °C and the use of a buffer, e.g. TRIS to maintain the pH from about 4.5 to 7 during the determination. The appropriate amount of benzidine derivative can be determined by routine experimentation as is known for peroxidase determinations, for example, it can range from 0.01 to 0.2 mg per ml of the test sample containing the peroxidase enzyme. In accordance with the invention, the phenylene derivative II is present during the peroxidase benzidine derivative reaction. It is used in color retarding amounts such that color development is retarded for a sufficient period of time to permit sharp end point determinations of the absence or presence of color, for example, it can be used in an amount that ranges from about 0.0005 to 0.01 mg per ml of test sample containing the peroxidase enzyme. Expressed another way, the phenylene derivative is present in an amount from about 0.1 to 10 weight percent of benzidine derivative I. The admixture of phenylene derivative II does not influence the color absorbance peak. The admixture of II to the reaction system I/H₂O₂/peroxidase results in a retardation of the onset of color development, the duration of which is directly proportional to the amount of II added and is inversely proportional to the amount of enzyme. During the retardation phase, color development is completely suppressed. After the onset of color development, its rate is the same as that observed without admixture of II under the same conditions. The visible range of the absorption spectrum remains qualitatively unchanged. The combination of the enzyme-dependent retardation of color development with a fixed reaction time for observation of color results in the desired ability to answer the passing of a threshold concentration by an unambiguous yes/no signal, in this case: colored/not colored. The invention may be further illustrated using as an example the combination of 3,3',5,5'- t etra m ethyl b enzidine (TMB) with hydroquinone for the determination of HRPO. With TMB the maximum absorption peak for the blue color is at 645 nm. This is not altered by the admixture of hydroquinone. However, admixture of hydroquinone to the reaction system TMB:H₂O₂:HRPO results in the retardation of the onset of color development in the entire visible range of the spectrum (700-300 nm), the duration of which is directly proportional to the amount of hydroquinone added and is inversely proportional to the amount of HRPO (Diagrams 1 and 2). During the retardation phase the color development is completely suppressed, after the onset of color development its rate is the same as that observed without addition of hydroquinone under the same conditions. The retardation in the onset of blue color development which is inversely proportional to the amount of HRPO in combination with a fixed reaction time for observance of color results in the desired ability to answer the passing of a threshold concentration by an unambiguous yes/no signal; in this case: blue/not blue. This is illustrated by the experiment shown in table 1 (the photometrically obtained absorbance values serve only for the exemplification of the effect which, according to the invention, allows the visual, non-instrumental evaluation of end dilution titrations). Starting with 1.1· 10⁻⁹ g HRPO per ml of substrate mixture two parallel dilution series with the dilution factors 1; 1.5; 2; 3; 4; 6; ... 128; 192 are prepared. By incubation with a substrate mixture containing only TMB as a proton donor in higher dilutions of the enzyme (A₆₄₅, 1cm ≦ 1) a gradually decreasing blue color is obtained which is directly proportional to the amount of HRPO. The identification of a dilution end point is not possible. If under the same conditions a substrate mixture is used which contains a small amount of hydroquinone, a dilution end point can be identified without doubt (highest dilutions with visually observable blue color: 1:8 after 30 minutes; 1:12 after 60 minutes). The threshold concentration of the enzyme may be adapted to the requirement by proper choice of the two parameters: Concentration of phenylene derivative II and reaction time to give suitable color development. A shift of the end point to an amount of enzyme lowered by the factor 1.5 would increase the reaction time necessary by at least the same factor. If reaction time and determination of end point are properly chosen (e.g. 30 min.), a deviation from this (e.g. reaction time between 24 and 36 min.) will lead to results different in one titer step at worst (factor: 1.5). Reaction time and end point determination will adequately be in a range where color development is sufficient, i.e. between 5 and 60 minutes. The unambiguity of the end point determination is exchanged for a small loss in the sensitivity of detection. But this is of no importance in many fields of application of EIA's with visual evaluation or may be compensated for by an adequate assay design. The combination of favourable properties which are offered by the principle of end dilution titration concerning low cost and high flexibility of instrumentation needed and wide measuring range with the virtues of enzyme-immuno-logical methods (specifity, sensitivity, universality of detection reaction) is very desirable. Results corresponding to those described here in detail for the combination TMB-hydroquinone are obtained with other combinations of benzidine derivatives I and phenylene derivatives II. This is true for the combination TMB/p-phenylendiamine TMB/m-phenylene diamine; TMB/1.2-dihydroxybenzene, o-tolidine/p-phenylene diamine; o-tolidine/m-phenylene diamine, o-tolidine/1.2-dihydroxy-benzene and o-tolidine/4-aminophenol as especially for the combinations TMB/hydroquinone; TMB/4-aminophenol TMB/o-phenylene diamine; o-tolidine/hydroquinone; o-tolidine/o-phenylendiamine; o-dianisidine/hydroquinone and benzidine/hydroquinone. This invention also relates to a reagent composition for the determination of peroxidase by end point dilution, containing a benzidine derivative of the general formula I in a sufficient amount to permit the determination of peroxidase activity by color formation and a phenylene derivative of the general formula II in a color retarding amount to prevent color formation for a sufficient period of time to permit a sharp end point determination. The reagent may contain the usual additives in the usual amounts including hydrogen peroxide or its adducts, e.g. urea or sodium borate-H₂O₂ adducts, in an amount sufficient to aid in color formation, buffers e.g. TRIS in amounts to maintain the pH range from 4.5 to 7 during the determination and chelating agents in chelating amounts for the chelating of heavy metals. Such reagent compositions for determination of peroxidase according to this invention may be distributed commercially as such or in the form of a kit. If they are distributed in the form of a kit, this kit contains the benzidine derivative, the phenylene derivative, usual additives, a peroxidase-labelled antigen or antibody conjugate and a reaction vessel or other solid support which is coated with antigen or antibody. The working instruction given in the following serves for the illustration of the invention without restricting it. EXAMPLE 1 Working Instructions (TMB/hydroquinone as an Example) Chromogen Stock Solutions 1. 10 g 3,3'5,5'-tetramethylbenzidine (analytical grade, free base) are dissolved in dimethylsulfoxide to give 100 ml. 2. 0.1 g hydroquinone is dissolved in dimethylsulfoxide to give 100 ml. Tris-Citrate-Buffer pH 5 12.1 9 tris(hydroxymethyl) aminomethane and 10.5 g citric acid-1-hydrate are dissolved in distilled water to give 1 liter. Substrate Mixture (to be prepared daily fresh) 50 ml tris-citrate buffer, 0.05 ml each of the two chromogen stock solutions and 0.1 ml 3% hydrogen peroxide are mixed. The reactions are started by the quick addition of 1 ml of the respective substrate mixture to 0.01 ml of the respective dilution of horse radish peroxidase. The evaluation of the color change is done after 5 to 60 minutes, preferably after 15 to 45 minutes. EXAMPLE II Kit for the Estimation of Antibodies Against Streptolysine-O in Human Sera The kit includes: a. Reagents for the determination of peroxidase activity by end dilution titration with visual evaluation, according to the invention: a solution of 10 mg/ml 3,3'5,5'-tetramethylbenzidine in dimethylsulfoxide a solution of 0.1 mg/ml hydroquinone in dimethylsulfoxide a solution of hydrogen peroxide (3%) a 0.1 mole per liter solution of tris-citrate-buffer, ph 5 b. A solution of a conjugate of horse radish peroxidase and rabbit IgG, directed against Human IgG. c. A microtiter plate coated with Streptolysine-O as an antigen-coated reaction vessel. d. A solution of 0.1 mole per liter tris-HCl buffer, pH 7,8 containing 0.1% Tween® 20; as a diluent for the serum samples to be tested and for antibody-peroxidase-conjugate b.

Claims: In a procedure for the determination of peroxidase activity by end dilution titration under peroxidase end point dilution titration determining conditions wherein a peroxidase is reacted with a benzidine derivative, the improvement comprising conducting said procedure utilizing as the benzidine derivative a benzidine derivative of the general formula (I) where R1 stands for a hydrogen atom, a halogen atom, an amino group, a (C₁-C₄) alkyl group, a (C₁-C₄) alkoxy group, a carboxy group, a sulfo group or a carboxypropyloxy group and where R2 stands for a hydrogen atom, a halogen atom, a (C₁-C₄) alkyl group, or a salt of this benzidine derivative, in the presence of a non-chromogenic or low-chromogenic phenylene derivative of the general formula (II) where R3 and R4 are equal or different and stand for a hydroxy group, an amino group, a (C₁-C₄) alkyl amino group or a benzyl amino group, to retard color development to permit a sharp dilution end point, and thereafter determining peroxidase activity. Procedure according to Claim 1, where R1 of the benzidine derivative I, stands for hydrogen, methyl, or methoxy and where R2 stands for hydrogen or methyl. Procedure according to Claim 1, where R1 and R2 of the benzidine derivative I, stand for methyl in ortho position to the respective amino groups. Procedure according to Claim 1, where the phenylene derivative II is hydroquinone, o-phenylene diamine, or 4-aminophenol. Procedure according to Claim 1, where the phenylene derivative II is hydroquinone. Procedure according to Claim 1, where the color is retarded for a period of 5 to 60 minutes. Procedure according to Claim 6, where the color is retarded for a period of 15 to 45 minutes. A reagent for determining peroxidase activity by end dilution titration comprising a benzidine derivative of the general formula (I) where R1 stands for a hydrogen atom, a halogen atom, an amino group, a (C₁-C₄) alkyl group, a (C₁-C₄) alkoxy group, a carboxy group, a sulfo group or a carboxypropyloxy group and where R2 stands for a hydrogen atom, a halogen atom, a (C₁-C₄) alkyl group, or a salt of this benzidine derivative and a phenylene derivative of the general formula (II) where R3 and R4 are equal or different and stand for a hydroxy group an amino group, a (C₁-C₄) alkyl amino group or a benzyl amino group. A kit for determining peroxidase activity by end dilution titration comprising (a) a benzidine derivative of the general formula (I) where R1 stands for a hydrogen atom, a halogen atom, an amino group, a (C₁-C₄) alkyl group, a (C₁-C₄) alkoxy group, a carboxy group, a sulfo group or a carboxypropyloxy group, where R2 stands for a hydrogen atom, a halogen atom, a (C₁-C₄) alkyl group, or a salt of this benzidine derivative, (b) a phenylene derivative of the general formula (II) where R3 and R4 are equal or different and stand for a hydroxy group an amino group, a (C₁-C₄) alkyl amino group or a benzyl amino group, (c) hydrogen peroxide or an adduct thereof, (d) a conjugate of horse radish peroxidase and an antigen or antibody, and (e) a solid support coated with an antigen or antibody which will react with the analyte to be determined.

Inventors: LENTFER, DIERCK, DR.
Applicants: BYK-SANGTEC DIAGNOSTICA GMBH & CO. KG
Application year: 1984
Main classification: C12Q1
IPC codes: C12Q1