US3616258A - Diagnostic product and process for the detection of niacin production by mycobacteria - Google Patents

Diagnostic product and process for the detection of niacin production by mycobacteria Download PDF

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US3616258A
US3616258A US834424A US3616258DA US3616258A US 3616258 A US3616258 A US 3616258A US 834424 A US834424 A US 834424A US 3616258D A US3616258D A US 3616258DA US 3616258 A US3616258 A US 3616258A
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zone
impregnated
contiguous
untreated area
reagent
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US834424A
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Donald P Kronish
William D Young Jr
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Warner Lambert Co LLC
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Warner Lambert Co LLC
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
    • 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
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/145555—Hetero-N

Definitions

  • a diagnostic product for the detection of niacin produced by human" Mycobacterium tuberculosis is prepared by impregnating a plurality of individual but separated zones on a paper strip with a series of reagents which include (1) an alkali metal salt of p-aminobenzoic acid; (2) sodium or potassium thiocyanate; (3) a crystalline acid such as citric, oxalic or malonic; and (4) chlorarriine-T.
  • the desired diagnostic test employing the impregnated paper strip is performed by bringing the strip into contact and in a sealed test tube with an extract of the culture to be tested. If niacin is present the reaction of the several reagents with the niacin leads to color formation as a positive test for the presence of niacin.
  • PATENTEDUET 26 I9
  • 3.616258 AREA l ZONE l3.5 mm A AREA 2 AREA 3 ZONE l2.5 mm B
  • AREA 4 AREA 5 ZONE 83 mm 5 mm c
  • AREA 6 AREA 7 ZONE l3.5 mm
  • D AREA 8 ZONE l2.5 mm
  • E AREA 9 ZONE 6.5 mm F m m INVENTOR.
  • the present invention provides a stable, sensitive diagnostic product for the detection by color development of niacin produced by human mycobacteria.
  • the novel diagnostic product is in the form of a strip of bibulous material impregnated with a series of reagents which react with niacin to produce a characteristic color. These diagnostic test strips are stable for 12 minutes at room temperature.
  • test strip of this invention it is possible to detect the presence of as little as 3.1 micrograms of niacin in a 0.6 ml. solution of an extract of the culture being tested. Since the test is performed in a sealed test tube, danger from toxic reagents is avoided.
  • the accompanying drawing is a diagrammatic representation of the bibulous test strip of the present invention.
  • the strip is divided longitudinally into a series of areas impregnated with a particular reagent and separated from each other by unimpregnated areas.
  • area 1 is impregnated with zone A Reagent, area 3 with Zone B Reagent, area 5 with Zone C Reagent and area 7 with Zone D Reagent; untreated areas 2, 4 and 6 separate the treated areas.
  • area 8 is impregnated with Zone E Hydrophobic Barrier and area 9 with Zone F Dye.
  • the diagnostic product of the present invention is prepared by impregnating a plurality of individual but separated zones of a suitable bibulous material, said zones being identified for convenience in relation to the specific reagent applied as Zones A, B, C and D with the following reagent media:
  • This is supplied in the form of an aqueous or an aqueous alcoholic solution of an alkali metal salt of paminobenzoic acid having a measured pH of from about 7.5 to 12;
  • Zone C A crystalline acid in dry form.
  • This may be applied as an aqueous solution of a crystalline acid such as citric, oxalic or malonic acid; and
  • an aqueous or saline extract of a culture of the organism to be tested is placed in a 13X 100 mm. test tube or similar container.
  • the reagent-impregnated diagnostic test strip is inserted into the extract fiuid and the container is immediately sealed. Capillary migration of reagents andgentle agitation, at room temperature, permits interaction of the reagents with any niacin present in the extract.
  • the development of a specific color reaction in about 15 to 20 minutes is a positive indication of the presence of niacin.
  • the color development reactions which take place when niacin is present are represented by:
  • the cyanogen chloride is formed by the reaction of the chloramine-T with the thiocyanate present on the test strip at an acid pH.
  • the diagnostic product of this invention is, as noted, stable for 12 months at room temperature.
  • the p-aminobenzoic acid reagent solution utilized for impregnating Zone A is prepared by dissolving from about 0.5 to 20 g. ofan alkali metal salt of p-aminobenzoic acid in about 20 to 100 ml., preferably about 30 to 50 ml. of distilled water, to which is then added a sufficient amount of a miscible organic solvent, such as ethanol, to bring the volume of the final solution to about 100 ml.
  • the concentration of the alcohol in the final solution can be from about 10 to percent but is preferably from about 40 to 60 percent by volume.
  • a sufficient amount of an alkaline agent which does not interfere with the diagnostic test is added to bring the pH of the final solution to a measured pH of from about 7.5 to 12, preferably about 9 to l2.
  • Suitable alkali metal salts of p-aminobenzoic acid which can be used include sodium and potassium salts; of these the sodium salt is preferred.
  • Suitable pH-modifying agents which do not interfere with the diagnostic test include such alkaline materials as sodium, potassium or ammonium hydroxide. Preferably about 0.lN sodium hydroxide solution is used.
  • the paminobenzoic acid salt employed is dissolved in approximately all of the distilled water to be used, the pH is adjusted to about 7.5 to 12, most preferably to about 10.5 to 10.7 and ethanol, the preferred alcohol solvent, is then added. The measured pH of this final solution falls between about-9 to l2.
  • the thiocyanate reagent solution applied to Zone B is prepared by dissolving from about 40 to 80 g., preferably about 50 to 70 g., of sodium or potassium thiocyanate in less than 100 ml. of distilled water and then bringing the volume of the solution to 100 ml. by adding further distilled water.
  • the acid reagent solution applied in Zone C is prepared by dissolving from about 30 to 70 g. preferably from about 40 to 60 g., of citric acid, oxalic acid or malonic acid, or a mixture of these acids, in distilled water and then adding sufficient additional distilled water to bring the volume of the final solution to 100 ml.
  • the chloramine-T reagent solution applied to form Zone D is prepared by dissolving from about 10 to 60 g. preferably from about 30 to 60 g., of chloramine-T in a lesser volume of distilled water and then adding further distilled water to bring the volume of the solution to 100 ml.
  • reagent solutions are applied to Zones A, B, C and D, respectively, of the bibulous material. If desired, they can be applied in several applications to the respective zone in order to apply and to provide the specific zones with a residue of the following amounts of reagent materials after the solvents in which they are applied have evaporated:
  • Color formulation as an indication of a positive result, will vary depending on the order of placement chosen for the several reagents. The following chart lists the operable combinations possible, along with the corresponding color reactions indicating a positive or negative result:
  • one end of the impregnated test strip may be coated with a hydrophobic material to serve as a means for handling the test strip while avoiding any contamination of the reagent zones.
  • a zone impregnated with a hydrophobic barrier composition hereinafter designated Zone E
  • Zone F a zone impregnated with a dye
  • This dyed zone can also be topcoated with a hydrophobic barrier composition such as that utilized for the coating of Zone E.
  • a composition which will prevent the leaching of the culture extract and reagent reactants upward to the end of the bibulous material or into the colored zone, if such a zone is present.
  • the barrier composition should, of course, be chemically inert in this system.
  • Substances which will form a waterproof barrier of this type include waxes, lacquers and plastic materials, particularly the colorless polymerized methyl methacrylate coating composition sold under the trade name KRYLON 150 CRYSTAL CLEAR, by Krylon, lnc., Norristown, Pa.
  • the KRYLON material is particularly preferred. it is supplied as a solution in a toluene vehicle and may be diluted for ease of application with additional toluene or other diluents such as ethyl, methyl, or propyl alcohol USP.
  • any suitable dye which will color the bibulous material sufficiently to distinguish the end of the diagnostic strip to be handled from the colorless reagent zones which are to be inserted into the culture extract may be used for the dye solution identified as Zone F.
  • dyes such as Sudan lV (Color Index No. 258 and Certification No. N218) or Sudan Red GGA in a suitable volatile solvent, i.e. xylene, may be used. Many other dyes are also suitable.
  • the preferred dye solution which is applied contains from about 0.05 to about 0.8 g., preferably from about 0.1 to about 0.5 g. of the dye in about lOO ml. of volatile solvent.
  • the bibulous materials suitable for use in producing the product of this invention are those materials which by means of capillary action are able to hold liquid.
  • Such materials include filter paper, felt, porous ceramic strips, woven or matted glass fiber and the like.
  • a particularly preferred form of paper for this purpose is Eaton-Dikeman No. 623 filter paper (70 lbs).
  • the diagnostic product is prepared by impregnating separate zones of Eaton-Dikeman No. 623 filter paper with successive applica tions of the reagents listed below until there has been deposited on the particular zone the preferred amounts of each reagent after solvent evaporation.
  • the preferred order in which the zones are applied is ABCDEF, as shown in the accompanying drawing, with each of zones ABCD being separated from each other by an intermediate untreated area 'and with Zones E and F immediately following and contiguous with Zone D without an untreated area in between.
  • citric acid 50 grams is dissolved in distilled water and the solution is brought to 100 ml. with additional distilled water. This material becomes cold as it dissolved and it must be warmed to room temperature.
  • chloramine-T 50 grams is dissolved in distilled water, with heating to 58-60 C. to insure complete dissolution. Sufficient distilled water is added to bring the volume to 100 ml, while maintaining the temperature at 58-60 C. to prevent recrystallization.
  • KRYLON 150 CRYSTAL CLEAR 85 ml. of KRYLON 150 CRYSTAL CLEAR is diluted with ml. of ethyl alcohol, USP.
  • Zone A reagent solution is applied to Area 1, twice one each side of the paper, allowing drying time between the first and second application to each side. This provides about 3.4 mg. of sodium p-aminobenzoic acid in each test strip.
  • Zone B reagent solution is applied to Area 3, twice on each side of the paper, allowing drying time between the first and second application to each side. This application provides about 9 mg. of potassium thiocyanate on each test strip. Care must be taken not to allow Zone A and Zone B reagent solutions to mix.
  • Zone C reagent solution is applied to Area 5, once on each side of the paper which deposits about 2.8 mg. of citric acid on the test strip.
  • Zone D reagent solution maintained at 58-60 C.
  • Zone B reagent solution is applied to Area 9, once on one side of the paper and allowed to dry.
  • Zone E reagent solution is applied to Areas 8 and 9, once on each side of the paper, coating both areas, and allowed to dry. After all areas of the paper have dried thoroughly, cut into Arinch (6.3 mm.) strips, each containing the nine areas impregnated with solutions herein described.
  • EXAMPLE 3 Use of the Diagnostic Composition Method of Use Place 0.6 ml. of a saline extract of the culture to be tested (Mycobacterium species) in a 13 X100 mm. or similar size tube. Add one diagnostic test strip prepared according to example 2, with Zone A (the end opposite the dyed marker zone) dipped into saline solution, the stopper immediately with a paraffin coated cork stopper. Allow to stand for 15 to minutes at room temperature with occasional gentle shaking. A positive test for niacin is indicated by the development of an orange to yellow color in the solution. A negative test is essentially colorless. Any color on the strip itself is disregarded. For disposal, one-half to 1 ml. of 10 percent aqueous NaOH should be added to each tube, restoppered, mixed and allowed to stand one-halfto 1 hour before autoclaving.
  • EXAMPLE 4 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence A, C, B, D; the same order and procedure is followed for Zones E and F as in examples 1 and 1
  • EXAMPLE 5 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence C, D, B, A; the same order and procedure is followed for Zones E and F as in examples 1 and 2.
  • EXAMPLE 8 The procedures of examples 1 and 2 are followed except that the reagent zones are applied to the paper in the sequence C, B, D, A; the same order is followed for Zones E and F, as in examples 1 and 2.
  • EXAMPLE 9 The procedure of example 3 is followed, using the test strip as prepared in example 5. As a positive indication of the presence of niacin, the strip becomes orange to pink and a yellow cross-reaction also appears on the test strip. The positive or negative color reactions of the test strips of examples 6, 7 and 8 can be found above.
  • a diagnostic composition for the positive detection of niacin production by mycobacteria which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein:
  • Zone A contains from about 0.2 to about 6 mg. of an alkali metal salt of p-aminobenzoic acid
  • Zone B contains from about 0.6 to about 20 mg. of sodiurn or potassium thiocyanate;
  • Zone C contains from about 0.2 to about 10 mg. of a crystalline acid selected from the group citric acid, oxalic acid and malonic acid; and
  • Zone D contains from about 1 to about 20 mg. of
  • zones being arranged in an order adjacent to each other which will give a positive reaction on contact with an aqueous or saline extract ofa niacin-producing culture.
  • Zone F A diagnostic composition according to claim 2, wherein an additional Zone F, impregnated with a dye solution and topcoated with a hydrophobic barrier composition is contiguous to Zone E.
  • a diagnostic composition for the positive detection of niacin production by mycobacteria which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein:
  • Zone A contains from about 1.0 to about 4 mg. of an alkaii metal salt of p-aminobenzoic acid;
  • Zone B contains from about 6.0 to about 13 mg. of sodium or potassium thiocyanate
  • Zone C contains from about 2.0 to about 3.0 mg. of a crystalline acid selected from the group citric acid, oxalic acid, or malonic acid; and
  • Zone D contains from about 2 to about mg. of
  • zones being arranged in an order adjacent to each other which will give a positive reaction on contact with an aqueous or salineextract of a niacin-producing culture.
  • a diagnostic composition according to claim 4, wherein an additional Zone E, positioned contiguous to the untreated area following the outermost reagent-impregnated zone, is impregnated with a chemically inert hydrophobic barrier composition comprising:
  • a diagnostic composition for the positive detection of niacin production by mycobacteria which comprises a bibulous material containing at least four reagent-impregnated zones, separated one from the other by an untreated area of the bibulous material, wherein:
  • Zone A contains about 3.4 mg. of the sodium saltof paminobenzoic acid
  • Zone B contains about 9 mg. of potassium thiocyanate
  • Zone C contains about 2.8 mg. ofcitric acid
  • Zone D contains about 8 mg. of chloramine-T', wherein the reagent-impregnated zones are positioned on the bibulous material in the sequence Zone A, Zone B, Zone C and Zone D, and wherein Zone A is contiguous only to the untreated area preceding Zone E; Zone B is contiguous only to the untreated area following Zone A and the untreated area preceding Zone C; Zone C is contiguous only to the untreated area foliowing Zone B and the untreated area preceding Zone D; Zone D is contiguous to the untreated area following Zone C.
  • Zone E positioned contiguous to Zone D, is im-v pregnated with a chemically inert hydrophobic barrier composition comprising about mg. of a methyl methacrylate coating composition and about 15 ml. of ethyl alcohol.
  • Zone C is contiguous only to the untreated area preceding Zone D
  • Zone D is a contiguous only to the untreated area following Zone C and the untreated area preceding Zone B
  • Zone B is contiguous only to the untreated area following Zone D and the untreated area preceding Zone A
  • Zone A is contiguous to the untreated area following Zone B.
  • a process for the detection of niacin production by mycobacteria comprising:

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  • Engineering & Computer Science (AREA)
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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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US834424A 1969-06-18 1969-06-18 Diagnostic product and process for the detection of niacin production by mycobacteria Expired - Lifetime US3616258A (en)

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US (1) US3616258A (enrdf_load_stackoverflow)
JP (1) JPS5031037B1 (enrdf_load_stackoverflow)
CA (1) CA927258A (enrdf_load_stackoverflow)
CH (1) CH545480A (enrdf_load_stackoverflow)
DE (1) DE2029822C3 (enrdf_load_stackoverflow)
FR (1) FR2050983A5 (enrdf_load_stackoverflow)
GB (1) GB1278036A (enrdf_load_stackoverflow)
SE (1) SE380043B (enrdf_load_stackoverflow)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3895914A (en) * 1972-02-04 1975-07-22 Orion Yhtymae Oy Means for isolation and detection of barbituric acid derivatives and glutethimide in biological fluids
US4066511A (en) * 1974-08-12 1978-01-03 Montagnon Paul A F Analytic device and method
US4168204A (en) * 1976-02-20 1979-09-18 Beatrice Foods Co. Stable freeze-dried Lowenstein-Jensen medium and a method for its preparation
US6287796B1 (en) * 1998-06-16 2001-09-11 Niadyne Inc Biochemical method to measure niacin status in a biological sample
US20080003629A1 (en) * 2006-04-07 2008-01-03 Morris Shayne K Device and method for detection of vitamins and nutritional minerals
CN103901193A (zh) * 2012-12-26 2014-07-02 深圳先进技术研究院 苏丹红检测免疫试纸及其制备方法
US20140287439A1 (en) * 2011-10-06 2014-09-25 Ingibio, Ltd. Method for manufacturing multiple-diagnosis membrane sensor by using screen printing

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6160743U (enrdf_load_stackoverflow) * 1984-09-21 1986-04-24

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3895914A (en) * 1972-02-04 1975-07-22 Orion Yhtymae Oy Means for isolation and detection of barbituric acid derivatives and glutethimide in biological fluids
US4066511A (en) * 1974-08-12 1978-01-03 Montagnon Paul A F Analytic device and method
US4168204A (en) * 1976-02-20 1979-09-18 Beatrice Foods Co. Stable freeze-dried Lowenstein-Jensen medium and a method for its preparation
US6287796B1 (en) * 1998-06-16 2001-09-11 Niadyne Inc Biochemical method to measure niacin status in a biological sample
US6428972B2 (en) 1998-06-16 2002-08-06 Niadyne, Inc. Biochemical method to measure niacin status in a biological sample
US20080003629A1 (en) * 2006-04-07 2008-01-03 Morris Shayne K Device and method for detection of vitamins and nutritional minerals
US20140287439A1 (en) * 2011-10-06 2014-09-25 Ingibio, Ltd. Method for manufacturing multiple-diagnosis membrane sensor by using screen printing
US9970933B2 (en) * 2011-10-06 2018-05-15 Ingibio, Ltd. Method for manufacturing multiple-diagnosis membrane sensor by using screen printing
CN103901193A (zh) * 2012-12-26 2014-07-02 深圳先进技术研究院 苏丹红检测免疫试纸及其制备方法

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FR2050983A5 (enrdf_load_stackoverflow) 1971-04-02
SE380043B (enrdf_load_stackoverflow) 1975-10-27
DE2029822C3 (de) 1975-04-17
CA927258A (en) 1973-05-29
GB1278036A (en) 1972-06-14
DE2029822A1 (de) 1971-01-28
DE2029822B2 (de) 1974-08-15
CH545480A (de) 1973-12-15
JPS5031037B1 (enrdf_load_stackoverflow) 1975-10-06

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