STABILIZED ENZYME SUBSTRATE SOLUTIONS

Abstract: A solution comprising a chromogen or substrate for use in an enzyme immunoassay, enzyme linked immunosorbent assay, immunoenzymometric assay or immunoperoxidase assays is stabilized to substantially reduce substrate drift. This is achieved by the addition of a chelating agent in an amount effective to substantially prevent nonenzymatic oxidation of the substrate or chromogen.

Description: STABILIZED ENZYME SUBSTRATE SOLUTIONS DESCRIPTION OF THE INVENTION: This invention relates to an improved enzyme immunoassay (EIA), enzyme linked immunosorbent assay (ELISA), immunoenzymometric assay or immunoperoxidase assay and pertains most specifically to a substrate solution for determining enzyme activity which contains ethyienediaminetetraacetic acid resulting in increased stability of the substrate solution and decreased substrate drift. Enzyme immunoassays, enzyme linked immunosorbent assays and immunoenzymometric assays involve the use of an enzyme, such as a peroxidase, as a label for the unknown in an assay procedure, and measurement of the enzyme activity as an indication of the amount of the unknown in the sample. Immunoperoxidase assays apply the same principles to microscopic examination of cultures or tissues. Such assays typically require the addition of the substrate for the enzyme after the immunoassay in order to determine the amount of "bound" enzyme. Peroxidases, such as horseradish peroxidase, require two substrates — a peroxide and a chromogen which affords a colored product upon oxidation. Typically, stock solutions of the peroxide and the chromogen are combined in the appropriate buffer to form a "working" substrate solution. With the usual chromogen, o-phenylenediamine, the working solution is quickly oxidized nonenzymatically resulting in an increase in color development even in the absence of peroxidase. Thus, the working solution must be used within 1/2 hour of preparation. A better chromogen, 3,3',5,5'-tetramethylbenzidine (TMB) has been developed. The working solution with TMB and peroxide also will be oxidized nonenzymatically, although more slowly. Thus the solution may be prepared one to two hours in advance of use. However, it has been noted that occasionally the working solution becomes colored very quickly, often within seconds. In such cases, the solution is not usable. This change in intensity of background color termed "substrate drift" greatly limits the utility of such substrate solution in enzyme immunoassay or enzyme linked immunosorbent assays. I have now found that such nonenzymatic oxidation and, hence, color development may be prevented by the addition of a chelating agent to the stock solutions from which the working solution is made. Experiments have indicated that such nonenzymatic oxidation, may be due to contamination by heavy metals. The addition of ferric ions to the working solution cause immediate generation of blue color. The addition of chelating agent prevents the unidentified heavy metals from catalizing oxidation increasing stability of the substrate solution and decreasing substrate drift. The enzyme-substrate system in which the present working solution is useful is any such system in which concentration or presence of the unknown is detected by enzymatic oxidation of the substrate. For example, the present invention can be used to determine peroxidase act vity of any enzyme which catalyzes the reaction of the chromogen with peroxide to form a colored compound. Although peroxidases, such as horseradish peroxidase, are among the more widely used of such enzymes, other peroxidases may also be used. "Chelating agent" as used in this invention means any compound in which a dicationic, tricationic or tetracationic metal is bound to two or more atoms or complexes with ligands containing more than one point of attachment. Chelating agents which can be used in the present invention include any chelating agent which binds heavy metals preventing nonenzymatic oxidation of the substrate solution without preventing enzymatic oxidation or the formation or detection of color. Preferred chelating agents are nontoxic and do not present a biohazard. These include, but are not limited to: ethyienediaminetetraacetic acid (EDTA) and other tetraacids, diethylenetriaminepentaacetic acid (DTPA) and other pentaacids, iminodiacetic acid and other derivatives, nitrilotriacetic acid and derivatives, succinic acid and other diacids, citric acid and other hydroxyacids and acetylacetone and other dicarbonyl compounds. Preferred chelating agents are EDTA and its derivatives, DTPA and its derivatives, iminodiacetic acid and its derivatives, and nitrilotriacetic acid and its derivatives. The most preferred is EDTA due to its solubility in the buffer of the stock and working solutions and its ability strongly to chelate a variety of metal ions at the pH of the working solution. Chelating agents which are not useful in the present invention are those which actually increase the rate of non-enzymatic oxidation of the chromogen or substrate, such as dip colinic acid. These chelating agents will be obvious to one skilled in the art without undue experimentation. The concentration of chelating agent in the working solutions will be dependent upon effective el mination of drift and minimization of interference with the enzymatic oxidation of the substrate. In general, it is desirable to minimize the concentration of chelating agent. Preferred concentrations are in the range of from about O.OlmM to about lOO M. The most preferred concentrations are in the range of from about 0.05mM to about lOmM. Substrates and chromogens with which the present invention is useful include those which are used in the detection of peroxidases. Chromogens which are useful include those which are well known in the art such as o-phenylenediamine, 2,2'-azinodi (3-ethyl) benzthiazoline-6-sulphonic acid (ABTS), dianisidine, dicarboxidine, TMB, diaminobenzidine. Preferred chromogens include any 3,3',5,5'-tetraalkylbenzidine in which the alkyl groups each contain from 1 to 5 carbon atoms; particularly useful are 3,3',5,5'- tetramethylbenzidine and 3,3',5,5'-tetraet_ylbenzidine. Acid salts such as the hydrochlorides also are useful. The amount of chromogen present in the substrate solution can vary over a considerable range, depending upon the identity and concentration of the peroxidase enzyme whose activity is to be measured; in general, the concentration of chromogen can vary from about 0.1 to about 10 M, preferably from about 1 to about 3mM. The amount of peroxide present also may vary, depending upon the amount of chromogen present, ranging from about 1 to about 20mM, but preferably it is from about 1 to about 6mM. Any of the usual peroxides such as hydrogen peroxide, urea peroxide, or the like can be employed in the substrate solution. It is also desirable to include a buffer in the solution, such as an acetate buffer, which maintains the pH from about 4 to about 7. The working solution is prepared by mixing a first stock solution containing a chromogen and a chelating agent with a second stock solution comprising an aqueous buffer solution containing the substrate and a chelating agent. The chelating agents of the two solutions may be the same or may differ. The chromogen solution can also comprise stabilizing or solubilizing agents. In the preferred peroxidase system, the working solution is prepared by mixing a first stock solution comprising an aqueous solution containing 5 to 50% N-methyl pyrrolidone by volume, 0.5 to ImM EDTA and 1 to 10 M 3,3',5,5'-tetraalkylbenzidine or an acid salt thereof with a second stock solution comprising an aqueous buffer solution containing 0.5 to ImM EDTA and 4 to 40 mM peroxide, and water if necessary to achieve the desired concentration. Stock solutions can be supplied in the form of a kit which contains in addition a supply of conventional stopping agent solution, antibody- or antigen-coated containers, and antigen or antibody standards, calibrators or controls. The determination of enzyme activity is carried out in the usual manner by incubating the substrate solution with the specimen containing the enzyme to develop a visible color. For quantitative determinations the reaction w ' ith the substrate solution is stopped after an established time by adding a conventional stopping agent such as, for example, an aqueous solution of water-soluble flouride, such as sodium fluoride, at pH from about 3 to about 6; or acids such as sulfuric acid, hydrochloric acid, etc. A soluble fluoride is preferred because it provides a stable blue color. The present invention can be employed with any of the usual enzyme immunoassay procedures, either homogeneous or heterogeneous assays, and either single- or double-antibody assays, and including enzyme linked immunosorbent assays, immunoenzymometric assays and immunoperoxidase assays. The following examples are intended to illustrate more fully the nature of the present invention without acting as a l mitation upon its scope. EXAMPLE I Aqueous substrate solutions were prepared with the following compositions: A. 7.5 M 3,3',5,5'-tetramethylbenzidine, 40% by volume N-methyl pyrrolidone. B. 5 n*1 hydrogen peroxide, 100 mM acetate. C. 7.5 mM 3,3',5,5'-tetramethylbenzidine, 40% by volume -4 N-methyl pyrrolidone, 10 M EDTA. -4 D. 5mM hydrogen peroxide, 100 mM sodium acetate, 10 M EDTA. The solutions were stored in glass at 4°C. Working substrate solutions with and without EDTA were prepared by combining one part of the respective TMB solution with 4 parts of the respective peroxide/acetate stock solution. The working solutions were stored in clear glass bottles at room temperature or at 4° to 6°C. At various intervals 200 μL (microliters) of the solution were acidified with 50μl l.ON H ^ SO, and the absorbance at 450 nm was measured in a spectrophotometer using water as a blank standard. The results are recorded in Table 1. Table 1 STABILITY OF WORKING SOLUTION AT ROOM TEMPERATURE Absorption at 450 nm Time After Preparation Without EDTA 10~ 4 M EDTA 0 0.013 0.010 17 hr 0.625 0.007 5 d 1.820 0.053 7 d 0.551 0.097 AT 4 to 6°C 0 0.010 0.010 2 hr 0.073 0.012 4 d 1.366 0.010 6 d .1.965 0.020 As can be seen from the data, the working substrate solution which contained EDTA was very stable for several days at 4 to 6°C and at room temperature. Without EDTA the nonenzymatic oxidation was so rapid that after a few hours the solution was no longer usable. The drop in absorbance at 7 days is probably due to the decomposition of the colored product occurring at a faster rate than it is formed. EXAMPLE II The effect of EDTA on enzyme activity was determined by running a horseradish peroxidase assay and varying the amount of horseradish peroxidase (lng/ml). 400 μl of the working solution of Example I, with and without EDTA, were added. The reaction was stopped after 10 minutes with 1 ml of 1 M H„S0. and the absorbances at 450 nm were determined as before. TABLE 2 ENZYME ACTIVITY Volume of Absorption at 450 nm HRP(μl) No EDTA 10" M EDTA 0 0.015 0.004 10 0.117 0.156 20 0.243 0.288 40 0.503 0.600 60 0.826 0.875 Results show that EDTA had very little effect on the enzyme activity. EXAMPLE III Enzyme immunoassays were performed using the EDTA stabilized TMB/peroxide system for quantitation of carcinoembryonic antigen (CEA). CEA standards were prepared containing 0, 2, 5, 10 and 25 ngm/ml of CEA in serum respectively. 100 μl of each standard were added along with 100 μl of 50 nM acetate pH 5.3 buffer to separate IgG (anti-CEA) coated microtiter wells. The assays were incubated for 2.0 hours at 37°C. Each well was washed three times with water. A conjugate was formed by covalently linking A'-CEA IgG to enzyme horseradish peroxidase (HRP) diluted in buffer with protein stabilizer. 200 μl of the conjugate was added to each well. The assays were further incubated for 1.0 hour at 37°C. The wells were washed with water as before and 200 μl of EDTA stabilized working solution prepared as in Example I was added to each well. The assays were incubated for 30 minutes at 20 to 23°C. The reaction was stopped by the addition of 50 μl per well of 0.1% NaF pH 3.5. The wells were then read in a microtiter spectrometer at 650 nm (See Table 3). TABLE 3 QUANTITATION OF CEA BY EIA CEA Concentration Ngm/ml Absorbance (650 nm) 0 0.037 2 0.142 5 0.311 10 0.625 25 1.58 EXAMPLE IV Working solutions were prepared as in Example I varying the -4 chelating aqent. The solutions with and without 10 M chelating agent were stored in clear glass bottles at room temperature. At various times 200 μl of the solution were used to determine absorbance at 650 nm. Stop solution (1.0 N H SO.) was not used. The results are shown in Table 4. TABLE 4 ' STABILITY OF WORKING SOLUTION AT ROOM TEMPERATURE Absorption at 650 nm Time after Preparation Chelating Agent (lO '4 Molar) 0 2 hr. 6 hr. 1 d 3 d NONE 0.018 0.063 0.145 0.158 0.845 EDTA 0.015 0.015 0.017 0.015 0.039 DTPA 0.015 0.019 0.021 0.015 0.042 Iminodiacetic Acid 0.014 0.020 0.024 0.043 0.102 Dipicolinic Acid 0.031 0.124 0.219 0.490 0.879 Citric Aci 0.020 0.020 0.024 0.050 0.053 The data shows that all of the chelating agents except dipicolinic acid prevented rapid non-enzymatic oxidation of the solution. Dipicolinic acid, on the other hand, increased non-enzymatic oxidation relative to the control and would not be suitable for use as a stabilizer. EXAMPLE V To test the effect of chelating agents on enzyme sensitivity, enzyme immunoassays for Hepatitis B surface antigen (HBsAg) were run using a TMB/Peroxide substrate system prepared with and without varying chelating agents. Controls were prepared using HBsAg diluted in buffer with 1% bovine serum albumin as a positive and human serum as a negative. 200 μl of control were added to anti-BHsAg coated microtiter wells. 200 μl of water was used for the blank. The wells were incubated for 2 hours at 37°C and then washed three times with water. 200 μl of a A'-BHsAg-HRP conjugate comprised of anti-HBsAg IgG covalently linked to horseradish peroxidase diluted in buffer with protein stabilizer was added to each well. The wells were incubated for one hour at 37°C and then washed as before. To each well was added 200 μl of working solution, with or without the chelating agent. The wells were incubated at 20-23°C for 30 minutes. The reaction was then stopped by the addition of .50 μ per well ' of 0.1% NaF pH 3.5. Absorbances were read in a microtiter spectrophotometer at 650 nm. The results are given in Table 5. TABLE 5 HBsAg ENZYME IMMUNOASSAY Chelating Agent HBsAg Controls Added to Substrate Blank Negative Positive None 0.042 ■ 0.013 1.057 EDTA 0.033 0.008 1.038 DTPA 0.030 0.008 1.035 Iminodiacetic Acid 0.032 0.028 1.022 Dipicolinic Acid 0.424 0.008 0.686 Citric Acid 0.033 0.011 1.026 Results suggest little effect on the assay sensitivity by most of the chela-ting agents. Dipicolinic acid appears to reduce assay sensitivity by about 50% and, therefore, is not acceptable for use in this invention.

Claims: ft WHAT IS CLAIMED IS: 1. A solution for use in an enzyme-imunoassay, enzyme i linked immunosorbent assay, immunoenzymometric assay or immunoperoxidase assay, in which a chromogen and a substrate are 5 reacted with an enzyme in an aqueous or mixed aqueous-organic buffer, which comprises a substrate or a chromogen and an effective amount of chelating agent to substantially prevent nonenzymatic oxidation of the substrate or chromogen. 2. The solution of Claim 1 wherein the substrate is 10 peroxide and the enzyme is peroxidase. 3. The solution of Claim 2 wherein the chelating agent is selected from the group consisting of ethyienediaminetetraacetic acid (EDTA) and other tetraacids, diethylenetriaminepentaacetic acid (DTPA) and other pentaacids, iminodiacetic acid and other 15 derivatives, nitrilotriacetic acid and derivatives, succinic acid and other diacids, citric acid and other hydroxyacids and acetylacetone and other dicarbonyl compounds. 4. The solution of Claim 2 wherein the chelating agent is selected from the group consisting of ethyienediaminetetraacetic 20 acid, diethylenetriaminepentaacetic acid, iminodiacetic acid and citric acid. 5. The solution of Claim 4 wherein the chelating agent is eth lenediaminetetraacetic acid. 6. The solution of Claim 2 wherein the concentration of 25 chelating agent is between from about O.OlmM to about lOOM. 7. The solution of Claim 6 wherein the concentration of chelating agent is between from about 0.05mM to about lOM. 8. The solution of Claim 2 wherein the chromogen is selected from the group consisting of o-phenylenediamine, 30 2,2'-azinodi(3-ethyl)benzthiazoline-6-sulphonic acid (ABTS;, dianisidine, dicarboxidine, diaminobenzidine, any 3,3', 5,5'-tetraalkylbenzidine in which the alkyl groups each contain from 1 to 5 carbon atoms, and the acid salts thereof. 9. The solution of Claim 8 wherein the chromogen is ,3',5,5'-tetrameth 1benzidine.

Inventors: THOMAS DAVID A, THOMAS, DAVID, A.
Applicants: TRAVENOL GENENTECH DIAGNOSTICS, TRAVENOL-GENENTECH DIAGNOSTICS
Application year: 1986
Main classification: C12Q1
IPC codes: C12Q1, G01N33