Abstract:
The method is directed to assaying for biological components in a sample comprising step (A) generating an oxidase substrate in the presence of an amphoteric surfactant and in the absence of ferrocyanide; (B) initially generating hydrogen peroxide through said oxidase reaction on said substrate of oxidase with subsequent detection of the generated hydrogen peroxide using peroxidase and a color developer capable of being oxidized in the presence of amphoteric surfactant and ferrocyanide; and (C) correlating the amount of color developed to the amount of biological components in the biological sample. Even when the biological component to be detected is present in a very small amount, the interference of bilirubin can be eliminated in assays of biological components in which peroxidase generated from an enzymatic reaction is detected using peroxidase and a color developer capable of being oxidized.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to an assay method for biological components which is not affected by the presence of bilirubin. More particularly, the present invention relates to an assay method for biological components which is carried out by detecting, using peroxidase and a color developer capable of being oxidized, hydrogen peroxide generated from an enzymatic reaction, and which is free from errors ascribed to bilirubin. 
     2. Description of Related Art 
     Enzymatic analyses employ enzymes as analytical reagents. They are superior to other assay methods relying on chemical reactions with respect to specificity. In addition, enzymatic reactions proceed under mild conditions. Because of these features, enzymatic analyses are easily automated, and therefore are widely used in the field of clinical chemistry. 
     In some cases, however, enzymatic analyses are interfered with various kinds of impurities in biological samples to an extent that cannot be neglected when precise assay is needed. In such cases, the target substance is not necessarily the only substance that is assayed. Particularly, it is known that bilirubin in biological samples affects colorimetric determination in which a suitable oxidizing enzyme is reacted with a substance to be analyzed and hydrogen peroxide generated is contacted with a reagent to develop color in the presence of peroxidase (&#34;Japanese Journal of Clinical Chemistry&#34;, vol 8, No. 1, 63-72 (1980)). Therefore, in the determination of trace components of living bodies, interference by bilirubin is a problem which cannot be ignored. 
     In order to avoid the effects by bilirubin, various methods have been developed, including a bilirubin oxidase method (&#34;Clinical Chemistry&#34;, 30/8, 1389-1392 (1984)), an aminopyrin method (&#34;Clinical Chemistry&#34;, 27/11, 1941-1942 (1980)), a potassium ferrocyanide method (&#34;Clinical Chemistry&#34;, 26(2), 227-231 (1980)), and an albumin-aminopyrin method (Japanese Patent Publication (kokoku) No. 4-61640). Recently, a method which employs potassium ferrocyanide and albumin in combination has also been reported (Japanese Patent Application Laid-open (kokai) No. 60-228963). 
     However, none of these prior methods is successful in completely eliminating interference by bilirubin, particularly when trace components are assayed. In addition, the reagents themselves have problems; i.e., they tend to become colored, or proteins contained therein are degraded. 
     In view of the foregoing, the inventors of the present invention carried out detailed studies and found that when an amphoteric surfactant is combined with conventional assays for biological components using ferrocyanides, interference by bilirubin can be remarkably eliminated even though the target biological component to be detected is present in only a very small amount, leading to completion of the present invention. 
     SUMMARY OF THE INVENTION 
     Accordingly, an object of the present invention is to provide an assay method for biological components in which hydrogen peroxide generated from an enzymatic reaction is detected with peroxidase and a color developer capable of being oxidized in the presence of an amphoteric surfactant and a ferrocyanide. 
     The above and other objects, features, and advantages of the present invention will become apparent from the following description. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows absorption curves in the range of 300 to 700 nm in which a surfactant is added (curve (b)) or is not added (curve (a)) to bilirubin. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     There is no particular limitation on the amphoteric surfactants which can be used in the present invention. For example, alkylimidazolium betaine, alkylbetaine, alkylamide betaine, alkylalanine, alkylamine oxide, and derivatives thereof can be used. The alkyl groups of these amphoteric surfactants each preferably have 6 to 20 carbon atoms. Specific examples of commercially available amphoteric surfactants include Anhitol 24B (alkylbetaine derivative, by Kao), Anhitol 20Y (alkylimidazolium betaine derivative, by Kao), Enagycol C-40H (alkylimidazolium betaine derivative, by Lion), Lipomin CH (alkylimidazolium betaine derivative, by Lion), Enagycol C-30B (alkylamide betaine derivative, by Lion), and Anhitol 20N (alkylamine oxide derivative, by Kao). It is preferred that the amphoteric surfactants be used from 0.01 to 10% by weight and particularly preferably from 0.1 to 5% by weight in the detection system. 
     The ferrocyanides used in the present invention are, for example, alkali metal ferrocyanides such as potassium ferrocyanide and sodium ferrocyanide. It is preferred that the ferrocyanides be used from 1 to 100 μM and particularly preferably from 5 to 50 μM in the detection system. 
     The color developers used in the present invention which can be oxidized are preferably combinations of 4-aminophenazone (particularly, 4-aminoantipyrine) and hydrogen donors such as phenols or anilines. Examples of the hydrogen-donating phenols include phenol and p-chlorophenol, and examples of the hydrogen-donating anilines include N-alkyl-N-sulfoalkyl-m-toluidine and N,N-dialkylaniline. 
     The assay method of the present invention is applicable to any colorimetry assay in which an enzyme is reacted with a biological component to generate hydrogen peroxide, and the hydrogen peroxide is reacted with a color developer capable of being oxidized in the presence of peroxidase. The biological components are components co-existing with bilirubin in living bodies, such as components in blood (whole blood, plasma, and serum) and urine. Specific examples of the biological components include triglyceride, glucose, phospholipids (lecithin, sphingomyelin, and lysolecithin), total cholesterol, free fatty acids, uric acid, cholinesterase, creatinine, and creatine. 
     The enzymes which are reacted with the biological components mentioned above are not particularly limited as long as they ultimately generate peroxide in one way or another. Specific examples of the enzymes include lipoprotein lipase, glycerokinase, glycerol-3-phosphate oxidase, glucose oxidase, phospholipase D, choline oxidase, cholesterol esterase, cholesterol oxidase, acyl CoA synthetase, acyl CoA oxidase, uricase, cholinesterase, and cholinoxidase. 
     Examples of enzymatic reaction systems in which the above enzymes are reacted are illustrated below. ##STR1## 
     Needless to say, in reactions in which a biological component is reacted with an enzyme as shown by the above schemes, coenzymes and substrates can be used as desired. 
     Upon completion of the enzymatic reaction for generating hydrogen peroxide and the subsequent reaction for detecting the hydrogen peroxide generated, the amount of the dyes generated is determined to precisely measure the amount of the target biological component. 
     EXAMPLES 
     The present invention will further be described by way of examples, which should not be construed as limiting the invention. 
     REFERENCE EXAMPLE 1 
     FIG. 1 shows an absorption curve of bilirubin (curve (a)), and an absorption curve of bilirubin to which an amphoteric surfactant is added (curve (b)). 
     In detail, curve (a) depicts absorption when 2.6 ml of a buffer was added to 70 μl of a bilirubin solution, whereas curve (b) depicts absorption when 2.6 ml of a buffer containing 0.39% by weight of laurylbetaine was added to 70 μl of a bilirubin solution, both in the range from 300 to 700 nm. 
     As is apparent from FIG. 1, the absorption curve of bilirubin (a) shifts to a peculiar and different absorption curve (b) when laurylbetaine is added to bilirubin. From this, it is inferred that the stereochemical structure of bilirubin is altered by the presence of an amphoteric surfactant, and as a result, it becomes difficult for it to serve as a substrate of peroxidase, canceling the competition with a color developer capable of being oxidized. In an assay method in which an amphoteric surfactant and a ferrocyanide are used in combination, these two produce synergical effects to enhance the effect of the invention. 
     EXAMPLE 1 
     The samples used were human sera to which bilirubin was added at various predetermined concentrations. The amount of uric acid in the samples was determined using reagents for assaying uric acid formulated as shown in Table 1, and the effect of bilirubin was investigated. 
     
                                           TABLE 1__________________________________________________________________________             Comp. Examples                      Invention Examples             1    2 3 1     2     3__________________________________________________________________________First Reagent2-(N-Morpholino)ethanesulfonic acid                   75 mMN-Ethyl-N-(2-hydroxy-3-sulfopropyl)                   0.75                      mMm-toluidinePeroxidase              3.0                      u/mlBovine serum albumin             0.1 wt %                  --                    --                      --    --    --Alkylbetaine.sup.1)             --   --                    --                      0.39 wt %                            --    --Alkylamide betaine.sup.2)             --   --                    --                      --    0.45 wt %                                  --Alkylimidazolium betaine.sup.3)             --   --                    --                      --    --    0.87 wt %Second reagent2-(N-Morpholino)ethanesulfonic acid                   75 mM4-Aminoantipyrine       0.75                      mMuricase                 0.6                      u/mlPotassium ferrocyanide             --   0.015 mM__________________________________________________________________________ In Table 1, the numerals are concentrations in reagents. .sup.1) Anhitol 24B (Kao) .sup.2) Enagycol C30B (Lion) .sup.3) Anhitol 20Y (Kao) 
    
     ASSAY METHOD 
     A first reagent (260 μl) was added to a sample (70 μl) and the mixture was stirred. The mixture was then heated at 37° C. for 5 minutes. Absorption at a wave length of 546 nm was measured using a blank (no reagent) as a control (absorption I). 
     Thereafter, a second reagent (130 μl) was further added to the mixture and heated at 37° C. for 5 minutes. Absorption at a wave length of 546 nm was measured using a blank (no reagent) as a control (absorption II). As the blanks, purified water was used. From the thus obtained absorptions I and II, absorption of the sample was calculated according to the following equation: ##EQU1## (R stands for &#34;reagent&#34;). 
     Uric acid solutions of known concentrations were also treated in a similar manner as described above, and their absorptions were calculated. The results were compared with the absorption of the sample, and the uric acid concentration of the sample was obtained. 
     RESULTS 
     The results are shown in Table 2. As is apparent from Table 2, conventional methods in which nothing was added (Comparative Example 1), potassium ferrocyanide alone was added (Comparative Example 2) or potassium ferrocyanide and bovine serum albumin were added in combination (Comparative Example 3) presented considerably low results of measurements as the amounts of bilirubin in the samples increased. By contrast, in the assay methods of the present invention in which potassium ferrocyanide and an amphoteric surfactant were used in combination (Invention Examples 1 to 3), interference by bilirubin was quite successfully eliminated. 
     
                       TABLE 2______________________________________Concentration      Measurements of uric acid (mg/dl)of bilirubin in      Comp. Examples                    Invention Examplessamples (mg/dl)      1      2       3    1     2     3______________________________________0          4.5    4.4     4.3  4.4   4.5   4.55          2.1    4.0     4.0  4.4   4.5   4.310         1.1    3.6     3.6  4.4   4.4   4.315         0.7    3.2     3.2  4.3   4.3   4.220         0.5    2.8     2.9  4.2   4.3   4.230         0.4    2.1     2.3  4.1   4.2   4.050         0.3    1.1     1.5  4.0   4.0   3.8______________________________________ 
    
     EXAMPLE 2 
     The procedure of Example 1 (measurements of uric acid) was repeated except that the various surfactants shown in Table 3 were used in place of the surfactants in Table 1, and 0.5% by weight of each surfactant was added to both first and second reagents. The performance of the surfactants in eliminating interference by bilirubin was compared. 
     The results are shown in Table 3. The standards for the evaluation of effect shown in Table 3 were as follows. 
     STANDARDS 
     The evaluation was based on the difference between the uric acid measurement obtained when the concentration of bilirubin in the sample was 0 mg/dl and that obtained when the concentration of bilirubin in the sample was 50 mg/dl. 
     
         ______________________________________  0 to 0.5:           A  0.6 to 1.0:           B  1.1 to 3.0:           C  3.1 or more:           D______________________________________ 
    
     As is apparent from Table 3, the amphoteric surfactant was remarkably superior to other types of surfactants in eliminating interference of bilirubin. 
     
                       TABLE 3______________________________________          Measurements of          uric acid (mg/dl)            bilirubin                     bilirubin                              Evaluation            conc. =  conc. =  ofSurfactants      0 mg/dl  50 mg/dl effect______________________________________(not added)      4.4      1.5      DAmphoteric surfactant:laurylbetaine    4.7      4.3      AAnionic surfactants:triethanolamine laurylsulfate            3931.1   1378.3   Dammonium laurylsulfate            could not be  D            measuredsodium alkylnaphthalene            3055.4   1528.9   Dsulfonatesodium polyoxyethylene            21.4     4.5      Dlaurylether sulfatesodium polyoxyethylene            4.5      1.7      Dalkylether sulfatetriethanolamine polyoxy-            14.7     0.5      Dethylene alkylether sulfatesodium polyoxyethylene            30.0     5.4      Dalkylphenylether sulfateCationic surfactant:Cetyltrimethylammonium            -5.1     -6.7     DchlorideNonionic surfactants:polyoxyethylene lauryl ether            4.4      3.2      Cpolyoxyethylene oleyl ether            4.3      1.0      Dpolyoxyethylene higher            50.3     45.2     Dalcohol etherpolyoxyethylene nonylphenyl            4.5      3.4      Cetherpolyoxyethylene derivative            4.4      3.2      Ctetraoleic acid  4.5      2.0      Cpolyoxyethylene sorbitol______________________________________ 
    
     EXAMPLE 3 
     The samples used were human sera to which bilirubin was added at various predetermined concentrations. In a similar manner to that described in Example 2, triglyceride was assayed using the reagents formulated as shown in Table 4, and the effect of bilirubin was investigated. 
     The results are shown in Table 5. 
     
                       TABLE 4______________________________________      Comp.      Example Invention Examples      4       4          5______________________________________First Reagents2-(N-Morpholino)-        200    mM     200  mM    200  mMethanesulfonic acidGlycerokinase        0.9    u/ml   0.9  u/ml  0.9  u/mlGlycero-3-phosphate        3.75   u/ml   3.75 u/ml  3.75 u/mloxidasePeroxidase   1.95   u/ml   1.95 u/ml  1.95 u/mlSodium 3,5-dimethoxy-        1.75   mM     1.75 mM    1.75 mMN-ethyl-N-(2-hydroxy-3-sulfopropyl)anilineAlkylbetaine.sup.1)        --            0.26 wt %  --Alkylimidazolium        --            --         0.29 wt %betaine.sup.2)Second Reagents2-(N-Morpholino)-        200    mM     200  mM    200  mMethanesulfonic acid4-Aminoantipyrine        1.5    mM     1.5  mM    1.5  mMLipoprotein lipase        900    u/ml   900  u/ml  900  u/mlAlkylbetaine.sup.1)        --            0.26 wt %  --Alkylimidazolium        --            --         0.29 wt %betaine.sup.2)Potassium    7.5    μM  7.5  μM 7.5  μMferrocyanide______________________________________ In Table 4, the numerals are concentrations in reagents. .sup.1) Anhitol 24B (Kao) .sup.2) Anhitol 20Y (Kao) 
    
     
                       TABLE 5______________________________________Bilirubinconcentration     Measurements of triglyceride (mg/dl)in samples     Comp. Ex.  Invention Ex.                            Invention Ex.(mg/dl)   4          4           5______________________________________0         93         93          9310        87         92          9220        71         92          9230        59         91          9350        46         92          90______________________________________ 
    
     As is apparent from Table 5, the conventional method (Comparative Example 4) gave low values of measurements as the amount of bilirubin in the sample increased, whereas the methods of the present invention (Invention Examples 4 and 5) in which potassium ferrocyanide and an amphoteric surfactant were used successfully eliminated interference of bilirubin. 
     EXAMPLE 4 
     The samples used were human sera to which bilirubin was added at various predetermined concentrations. In a similar manner to that described in Example 2, total cholesterol was assayed using the reagents formulated as shown in Table 6, and the effect of bilirubin was investigated. 
     The results are shown in Table 7. 
     
                       TABLE 6______________________________________      Comp.      Example Invention Examples      5       6          7______________________________________First ReagentsPotassium    60     mM     60   mM    60   mMhydrogenphthalate4-Aminoantipyrine        2.3    mM     2.3  mM    2.3  mMCholesterol esterase        0.3    u/ml   0.3  u/ml  0.3  u/mlPeroxidase   4.5    u/ml   4.5  u/ml  4.5  u/mlAlkylbetaine.sup.1)        --            0.26 wt %  --Alkylamine   --            --         0.53 wt %oxide.sup.2)Second ReagentsPotassium    60     mM     60   mM    60   mMSodium 3,5-dimethoxy-        0.6    mM     0.6  mM    0.6  mmN-ethyl-N-(2-hydroxy-3-sulfopropyl)anilineCholesterol esterase        0.7    u/ml   0.7  u/ml  0.7  u/mlAlkylbetaine.sup.1)        --            0.26 wt %  --Alkylamine   --            --         0.53 wt %oxide.sup.2)Potassium    7.5    μM  7.5  μM 7.5  μMferrocyanide______________________________________ In Table 6, the numerals are concentrations in reagents. .sup.1) Anhitol 24B (Kao) .sup.2) Anhitol 20N (Kao) 
    
     
                       TABLE 7______________________________________Bilirubinconcentration     Measurements of T. cholesterol (mg/dl)in samples     Comp. Ex.  Invention Ex.                            Invention Ex.(mg/dl)   5          6           7______________________________________0         169        168         16810        154        165         16615        148        167         16920        142        166         16530        132        166         16950        110        166         166______________________________________ 
    
     As is apparent from Table 7, the conventional method (Comparative Example 5) gave low values of measurements as the amount of bilirubin in the sample increased, whereas the methods of the present invention (Invention Examples 6 and 7) in which potassium ferrocyanide and an amphoteric surfactant were used successfully eliminated interference of bilirubin. 
     According to the assay method of the present invention, even when the biological component to be detected is present in a trace amount, the interference of bilirubin can be effectively eliminated in assays of biological components in which peroxidase generated from an enzymatic reaction is detected using peroxidase and a color developer capable of being oxidized.