Classifications

• • 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
• • 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
  • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
  • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/904—Oxidation - reduction indicators

Definitions

• Wolk Assistant Examiner-Arnold Turk Attorney, Agent, or FirmBurgess, 'Dinklage & Sprung ABSTRACT Test strips are provided for detecting even small amounts of blood or other peroxidatively active substances in body fluids; the strips comprising a carrier containing a hydroperoxide, a chromogen, and as an activator, a compound of the formula i'cdc wherein R is a hydrogen atom or a methyl radical and benzene and/or pyridine rings are fused on at least one of the positions indicated with c, (d,e), f and g, provided that two adjacent rings must not simultaneously each contain a cyclic nitrogen atom, and wherein the.
• aromatic compounds (1) apart from the positions indicated by H and R, can be substituted by lower alkyl radicals which together can also form a hydroaromatic ring.
• hemorrhages are caused, for example, by tumors, ulcers .and inflammations of the corresponding organsQFurthermore, free hemoglobin can also occur in the urine and plasma due to the influence of certain hemolytic toxins in the blood.
• Bloodand hemoglobin are peroxidateactive, i.e., they liberate oxygen from hydroperoxides and transfer it to certain acceptors. Other peroxidateactive, substances occur in leukocytes and bacteria. The detection of these substances is important for the diagnosis of diseases and infections of the kidney and urinary tract.
• Myoglobin which is also perioxidateactive, is found in the urine, for example, after a cardiac infarct. Furthermore, blood occurs especially frequently in the urine when calculi are present in the bladder or kidneys.
• Rapid tests are usually absorbent carriers, preferably papers, which have'been impregnated with all of the reagents necessary for the detection reaction. After simply dipping them into a body fluid, they show a color reaction. Because of the great importance which rapid tests have recently achieved, they have been developed in various ways for the detection of blood in body flu.- ids.
• German Pat. No. 1,242,905 describes test papers which, as activating additives, contain certain quinoline derivatives, preferably quinine. lt is certainly asserted that the sensitivity can be considerably increased with the mentioned additives but, acccording to our own investigations, it is practically impossible also to detect leukocytes and bacteria with the test strips improved in this manner.
• the instant invention provides test strips for the detection of peroxidate-active substances with a hitherto unachievably high sensitivity.
• test 5 strips comprising as the activator, an aromatic compound of the general formula:
• R is a hydrogen atom or a methyl radical and benzene and/or pyridine rings are fused on at least one of the positions indicated with c, (d,e), f and 3, provided that two adjacent rings must not simultaneously each contain a cyclic nitrogen atom, and wherein the aromatic compounds (1), apart from the positions indi cated by H and R can be substituted by lower alkyl radicals which together can also form a hydroaromatic ring.
• R is a hydrogen atom.
• R is a hydrogen atom.
• alkyl radicals there are to be understood alkyl radicals containing up to four carbon atoms and, when two alkyl radicals together form a hydroaromatic ring, 5- and 6- membered rings are preferred.
• the compounds of general formula (I) do not increase the sensitivity of peroxidases of vegetable origin, for example horseradish peroxidase, but act specifically on peroxidate-active substances of human and animal origin. It is thus possible selectively to detect leukocytes, blood and blood components and bacteria in feces or in vomit in the presence of vegetable peroxidases. In this case, myoglobin is detected with about the same degree of sensitivity as hemoglobin.
• the sensitivity of the detection reaction is increased by some of the compounds of general formula (l) to such an extent that even in urine it is possible to detect individual erythrocytes which are visible on the test paper as colored dots.
• a test paper with which it is still possible clearly to detect 5 erythrocytes per mm urine. This corresponds to a blood dilution of l:l,000,000.
• the sensitivity can be further modified by alkyl substitution; thus, for example, by means of a methyl substituent in a a-position to a cyclic nitrogen atom, the activity is somewhat reduced, whereas otherwise it usually leads to an increase of the activation.
• the sensitivity limit of a test strip which contains a weak activator for example 2, 8- dimethyl-l,7-phenanthroline, is about erythrocytes/mm urine.
• the activation agents according to the present invention can be used in amounts of about 0.05-1.0 percent, preferably of 0.2-0.5 percent, per 100 ml. impregnation solution.
• an organic hydroperoxide an oxidation indicator (chromogen), a buffer and a surface-active agent, as well as, if desired, a phosphoramide, for example phosphoric acid trimorpholide, for stabilization, as well as other conventional adjuvants.
• hydroperoxides there can be used, for example, cumol hydroperoxide or 2,5-dimethyl-hexane-2,5- dihydroperoxide, and as indicators those of the benzidine series, for example, o-tolidine, or those of the heterocyclic azines series, for example bis-(N-ethylquinol-2-one)-azine or (N-methylbenzthiazol-2-0ne)- (l-ethyl-3-phenyl-5-methyltriazol-2-one)-azine (see German Pat. No. 1,648,840).
• benzidine series for example, o-tolidine
• heterocyclic azines series for example bis-(N-ethylquinol-2-one)-azine or (N-methylbenzthiazol-2-0ne)- (l-ethyl-3-phenyl-5-methyltriazol-2-one)-azine (see German Pat. No. 1,648,840).
• the indicators can be used in amounts of from 0.05-5 g., preferably of 0.2-l .0 g., per ml. of impregnation solution.
• buffer there can be used, for example, a citrate, phosphate, phthalate or succinate buffer, the pH value and capacity being so chosen that, after dipping the test strip into a body fluid, a pH value of 4-7, preferably of 5-6, is obtained thereon.
• test strips due to the relatively large amounts of water-soluble substances present therein, could tend to bleed, it is of practical importance to add a thickening agent to the formulation, for example methyl cellulose and, in particular, gelatine, preferably in an amount of about 0.5-5 g. per I00 ml.
• a thickening agent for example methyl cellulose and, in particular, gelatine, preferably in an amount of about 0.5-5 g. per I00 ml.
• wetting agent there is preferably used a longchained organic sulphate or sulphonate, for example sodium dodecyl-benzene sulphonate, dioctyl sodium sulphosuccinate or sodium lauryl sulphate, which, as is known, stabilizes radical cations, such asoxidized otolidine.
• the wetting agents can be added to the impregnation solution in amounts of 0.5 to 5 percent, preferably of l 3 percent.
• absorbent carriers for example filter paper, cellulose or synthetic resin fleeces
• solutions of the reagents in readily volatile solvents This is preferably carried out in two separate steps. First, impregnation is carried out with a solution which contains a hydroperoxide, wetting agent, buffer and optionally a thickening agent. Thereafter, impregnation is carried out with a solution of an indicator and of an activator of general formula (I).
• test strips according to the present invention are,
• Solution 1 1.2M citrate buffer, pH 5.25 35.0 ml. ethylenediamine-tetraacetic acid, disodium salt 0.l g. dioctyl sodium sulphosuccinate 2.0 g. 2,5-dimethylhexane-2,S-dihydroperoxide (about 70%) 1.6 g. phosphoric acid trimorpholide l2.7 g. ethanol 30.0 ml. distilled water ad 100.0 ml.
• a white test paper is obtained which, upon dipping into a blood-containing urine, becomes green colored after about 5 to seconds. If the'erythrocytes are intact, then the paper is green flecked. lf hemolysis has taken place or if free hemoglobin is present in the urine, then the paper becomes uniformly green colored.
• the sensitivity is about 5 erythrocytes/mm or the corresponding amount of hemoglobin. A smaller number of intact erythrocytes can, under certain circumstances, still bring about individual green dots on the test paper. The sensitivity with regard to myoglobin corresponds to that for hemoglobin.
• Leukocytes and bacteria are also detected when intact, by flecking, or when lysed, by a uniform coloration.
• test paper obtained is about ten times less sensitive than the test papers according to Examples 1 and 2 (about 50-100 erythrocytes/mm in 3060 seconds).
• Test papers of similar sensitivity are obtained when, instead of 3-methylbenzo [f] quinoline, there is used an equimolar amount of one of the'following activators: 1,3-dimethylbenzo [f] quinoline; 2,4-dimethylbenzo [g] quinoline; 6-methylphenanthridine; 3,8-dimethyl- 4,7-phenanthroline; 2,8-dimethyl-l,7-phenanthroline; or 2,7-dimethyll ,o-anthrazoline.
• Test strip for the detection of peroxidatively-active substances in body fluids, comprising a carrier containing a hydroperoxide, at least one chromogen and, as an activator, a compound of the formula wherein R is hydrogen or methyl; and benzene and/or pyridine ririgs are fused on at least one of the positions indicated with c, (d,e), f and g, provided that two adjacent rings must not simultaneously contain a cyclic nitrogen atom, and wherein said compound can be substituted, other than in the positions indicated by H and R by lower alkyl radicals, which radicals together can also form a hydroaromatic ring.
• Test strip as claimed in claim 1, wherein the carrier is an absorbent material impregnated with the reagents.
• Test strip as claimed in claim 1, wherein the carrier is a water-stable film with the reagents incorporated therein.
• Test strip as claimed in claim 1, wherein the reagent solution used for the production thereof contains 0.2-0.5 percent of activator per 100 ml. of solution.
• Test strip as claimed in claim 1, wherein the reagent solution used for the production thereof contains 0.05-5 g. of chromogen per 100 ml. of solution.
• Test strip as claimed in claim 1, wherein the reagent solution used for the production thereof contains a buffer and or a complex former, a thickener or a wetting agent.
• Method of detecting small amounts of blood in a body fluid comprises contacting a test sample of the body fluid with a test strip as claimed in .claim 1 and observing the color formation thereon as an indication of the absence or presence of blood in said saple.
• Method of detecting small amounts of peroxidatively active substances in a body fluid comprises contacting a test sample of the body fluid with a test strip as claimed in claim 1 and observing the color formation thereon as an indication of the absence or presence of peroxidatively active substances in said sample.

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• Chemical & Material Sciences (AREA)
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• Genetics & Genomics (AREA)
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• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)

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Cited By (38)

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Citations (2)

• 1972
  • 1972-07-18 DE DE2235152A patent/DE2235152C2/de not_active Expired
• 1973
  • 1973-07-03 US US00376248A patent/US3853472A/en not_active Expired - Lifetime
  • 1973-07-11 CA CA176,154A patent/CA988011A/en not_active Expired
  • 1973-07-12 SE SE7309791A patent/SE388693B/xx unknown
  • 1973-07-12 AR AR294043A patent/AR195135A1/es active
  • 1973-07-13 IT IT26612/73A patent/IT994949B/it active
  • 1973-07-13 NL NL737309779A patent/NL152085B/xx not_active IP Right Cessation
  • 1973-07-13 FI FI2240/73A patent/FI53511C/fi active
  • 1973-07-16 CH CH1034773A patent/CH579275A5/xx not_active IP Right Cessation
  • 1973-07-16 FR FR7325947A patent/FR2198643A5/fr not_active Expired
  • 1973-07-16 DD DD172306A patent/DD105065A5/xx unknown
  • 1973-07-16 ZA ZA734810A patent/ZA734810B/xx unknown
  • 1973-07-16 GB GB3373173A patent/GB1390900A/en not_active Expired
  • 1973-07-17 AT AT628773A patent/AT326276B/de active
  • 1973-07-17 HU HUBO1451A patent/HU166368B/hu unknown
  • 1973-07-18 JP JP48082405A patent/JPS583680B2/ja not_active Expired
  • 1973-07-18 AU AU58263/73A patent/AU468582B2/en not_active Expired
• 1978
  • 1978-02-21 BE BE185336A patent/BE864154Q/xx not_active IP Right Cessation
  • 1978-04-13 HK HK205/78A patent/HK20578A/xx unknown

Patent Citations (2)

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