Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase

Definitions

• 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.
• EIA enzyme immunoassay
• ELISA enzyme linked immunosorbent assay
• immunoenzymometric assay immunoperoxidase assay
• 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.
• the working 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.
• TMB 3,3',5,5'-tetramethylbenzidine
• 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.
• peroxidases such as horseradish peroxidase
• 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.
• EDTA ethyienediaminetetraacetic acid
• DTPA diethylenetriaminepentaacetic acid
• iminodiacetic acid and other derivatives 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.
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• Aqueous substrate solutions were prepared with the following compositions:
• 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.
• 200 ⁇ L (microliters) of the solution were acidified with 50 ⁇ l 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.
• 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.
• 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 ⁇ l 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 HdonS0. and the absorbances at 450 nm were determined as before.
• 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 ⁇ l of each standard were added along with 100 ⁇ l 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.
• HRP horseradish peroxidase
• 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.
• HBsAg Hepatitis B surface antigen
• 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 ⁇ l of control were added to anti-BHsAg coated microtiter wells. 200 ⁇ l of water was used for the blank. The wells were incubated for 2 hours at 37°C and then washed three times with water.

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• 1986
  • 1986-02-04 JP JP61501091A patent/JPS62502653A/ja active Pending
  • 1986-02-04 WO PCT/US1986/000229 patent/WO1986004610A1/en not_active Application Discontinuation
  • 1986-02-04 EP EP86901252A patent/EP0213156A1/de not_active Withdrawn
  • 1986-02-11 ZA ZA86983A patent/ZA86983B/xx unknown
  • 1986-10-01 FI FI863968A patent/FI863968A/fi not_active Application Discontinuation

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