EP0267952A4 - Testreagenzien und verfaren zur bestimmung von okkultem blut in fäkalien. - Google Patents

Testreagenzien und verfaren zur bestimmung von okkultem blut in fäkalien.

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Publication number
EP0267952A4
EP0267952A4 EP19870903812 EP87903812A EP0267952A4 EP 0267952 A4 EP0267952 A4 EP 0267952A4 EP 19870903812 EP19870903812 EP 19870903812 EP 87903812 A EP87903812 A EP 87903812A EP 0267952 A4 EP0267952 A4 EP 0267952A4
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solvent
mixture
iron
hydroperoxide
approximately
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EP19870903812
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French (fr)
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EP0267952A1 (de
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Paul J Lawrence
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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
    • C12Q1/26—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 is in the field of fecal occult blood tests (FOBT) . More particularly it relates to improved FOBT that provide a reduced incidence of false results and/or greater sensitivity and specificity and/or are easier to perform.
  • FOBT fecal occult blood tests
  • FOBT are commonly used clinically to detect occult blood loss from gastrointestinal (GI) lesions.
  • GI gastrointestinal
  • carcinoma of the colon and rectum is the most serious cancer in the U.S. and second only to lung cancer in causing death—approximately 100.000 new cases and 50.000 deaths annually.
  • colorectal cancer is slowly progressive with a long asymptomatic period. t provides an ideal opportunity for early detection and successful therapy.
  • FOBT are a rational attempt at early diagnosis because the colorectal lesions frequently bleed, and routine noninvasive testing is possible.
  • hospitals and physicians very often utilize FOBT to detect or monitor GI lesions resulting from disease, injury, surgery, and other causes.
  • Guaiac is a complex plant extract containing the leuco dye, alpha guaiaconic acid.
  • Leuco dyes are oxidized by hydroperoxides in the presence of catalyst to form a blue color: hydroperoxide
  • hemoglobin is an efficient catalyst (pseudoperoxidase) . feces may be tested for occult blood using a leuco dye/hydroperoxide reagent. Nonetheless, the procedure remained very poorly utilized because of the disagreeable nature of the test and physicians were largely denied this very useful information.
  • U.S. Patent No. 3.996.006 describes a FOBT technique that popularized the guaiac-based test for occult blood in feces. It employs a slide having a sheet of guaiac-impregnated paper between a front panel and a rear panel with openings in the panels and pivotal flaps to cover the openings. A fecal specimen is placed on the paper through the opening in the front panel and that panel is closed. The rear panel is then opened and a hydrogen peroxide developer is placed on the paper via the opening in the rear panel. If blood is present in the specimen, the paper will turn blue. A commercial embodiment of this test, called the HEMOCCULT® test, is widely used in hospitals and physicians 1 offices.
  • the sensitivity limitation is due partly to (1) the fact .that hemoglobin in many specimens is degraded to derivatives that exhibit little or no peroxidative activity. (2) degradation of peroxidatively active hemoproteins by the hydroperoxide reagent used in the test and (3) the relative insolubility of the degraded products (i.e.. iron prot ⁇ porphyrins such as heme and he in) in the reagents used in the test.
  • Sensitivity limitations may cause false negative results.
  • the specificity limitation is probably due to the response of the test to plant peroxidases and/or iron or copper in the specimens or the environment in which the test is run. Specificity limitations lead to false positive results.
  • U.S. Patent No..4.333,734 describes a variation in the guaiac-based FOBT that is intended to reduce the incidence of false positive results due to the presence of plant peroxidases in the specimen. It includes a peroxidase denaturing agent such as urea or guanidine hydrochloride together with a metal chelating agent to sequester calcium and magnesium ions that are essential to peroxidase activity. The denaturant and the chelating agent are formulated with the guaiac.
  • a peroxidase denaturing agent such as urea or guanidine hydrochloride
  • a metal chelating agent to sequester calcium and magnesium ions that are essential to peroxidase activity.
  • the denaturant and the chelating agent are formulated with the guaiac.
  • U.S. Patent No. 4.071.317 relates to using polar solvents such as dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) to stabilize mixtures of organic hydroperoxides and leuco dyes that are used in FOBT.
  • polar solvents such as dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF)
  • the solvent is formulated in minor proportions with the hydroperoxide and leuco dye.
  • This solution is applied to a solid matrix and the matrix is dried prior to use in testing.
  • DMSO dimethyl sulfoxide
  • DMF dimethyl formamide
  • Biochem J (1979) 179_: 281-289 indicates that hemin occurs in its monomeric form in mixtures of DMSO and water that contain in excess of about 35% (v/v) DMSO.
  • Siochem J (1968) 108: 131-136 discusses the solubility of nitrogenous ligand-alkaline hematin complexes.
  • Biochimica et Biophysica Acta (1977) 498: 205-214 describes the use of various water-soluble polymers such as polyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone. and polystyrene sulfonate to dissolve aggregates of ferroheme and protoporphyrin in alkaline aqueous media.
  • various water-soluble polymers such as polyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone. and polystyrene sulfonate to dissolve aggregates of ferroheme and protoporphyrin in alkaline aqueous media.
  • An object of the present invention is to reduce the incidence of incorrect results (both false positive and false negative) in leuco dye-based FOBT. This is achieved by applying the hydroperoxide or both the hydroperoxide and leuco dye to the specimen in a solution that uses a specific solvent system that ⁇ dissolves iron protoporphyrins.
  • One aspect of the invention is a fecal occult blood test method comprising contacting a fecal specimen on. a solid test matrix with a leuco dye and a hydrop ⁇ oxide, wherein the hydroperoxide or the hydroperoxide and the leuco dye are applied to the specimen in solution in a solvent comprising at least 50% by volume of a solvent for iron protoporphyrins.
  • the solvent for iron protoporphyrins comprises a mixture of a neutralized amine and an organic co-solvent, whereby iron protoporphyrins in the specimen are dissolved.
  • the solvent for iron protoporphyrins comprises (a) an amide of the formula
  • R . R . and R are the same or different and represent hydrogen, lower alkyl. phenyl or benzyl.
  • R may be linked with R or R to form a 5- or 6-membered heterocycle
  • O 0 -S- or -S- 0 and R 1 and R2 are the same or different and are lower alkyl. phenyl. benzyl, or. when X is
  • R 1 and R2 may be linked to form a 5 or 6 membered heterocycle
  • Still another aspect of the invention is an FOBT developer composition for use in a fecal occult blood test which uses a leuco dye indicator, the developer composition comprising a solution of a hydroperoxide in an iron protoporphyrin solvent, characterized in that the iron protoporphyrin solvent comprises a mixture of a neutralized amine and an organic co-solvent.
  • an FOBT developer composition including an iron protoporphyrin solvent selected from (a) through (f) above.
  • a complete reagent composition for use in a fecal occult blood test comprising a solution of a leuco dye and a hydroperoxide in an iron protoporphyrin solvent, characterized in that the iron protoporphyrin solvent comprises either a mixture of a neutralized amine and an organic co-solvent, or is defined by (a) through (f) as outlined above.
  • he ⁇ toprotein is intended to include hemoglobin and derivatives of hemoglobin such as he e and hemin that have the ability (particularly in their monomeric form) to catalyze the transfer of oxygen from a hydroperoxide to a leuco dye to cause the leuco dye to be oxidized and thereby produce a detectable response. Such ability is sometimes referred to herein as "peroxidative activity".
  • leuco dye is not intended to be limited to a particular chemical species or genus but is intended to encompass indicators that produce a detectable response, typically a color change that is visible to the naked eye, when oxidized in the presence of a hemoprotein.
  • leuco dyes examples include guaiac, benzidine, o-tolidine. cresol, catechol. 3.3' .5,5'-tetramethylbenzidine. p-tolidine. beta- naphthol. pyrogallol, o-phenylenediamine. leuco malachite green, 3-amino ethylcarbazole. 4-amino antipyrine, phenol, and 2.2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid (ABTS).
  • ABTS 2.2'-azino-di-(3-ethylbenzyl) azoline sulfonic acid
  • chromagen mixture and hydroperoxide For long-term stability, it is preferred to keep the chromagen mixture and hydroperoxide separate. For instance, they might be packaged in separate containers adapted for simultaneous dispensing, such as a double-barreled syringe with a common outlet nozzle.
  • Another format is to employ the chromage ⁇ in solution as part of a complete reagent (when long-term stability is not involved) .
  • the complete reagent contains, in addition to the mixture and the solvent, a hydroperoxide and. optionally, other additives such as hemoprotein solubilizing agents, stabilizers, vegetable peroxidase inhibitors, iron chelators. accelerators, and buffers. Such additives may. of course, be impregnated into the solid test matrix in the other test formats.
  • the chromagen mixture of .guaiac and ABTS produces a color response that is greater than the sum of the responses that would be expected from the individual components. In addition to enhanced color intensity, the color is spread more evenly than the observed in current (e.g.. HEMOCCULT® test) FOBT, is more stable (i.e., long-lasting), and is more reproducible.
  • the weight ratio of guaiac to ABTS in the mixture will be in the range of 1:5 to 5:1, preferably approximately 1:1.
  • the chromagen mixture is formulated as a complete reagent with a peroxide and/or in a solvent for iron protoporphyrins such as DMSO, it is preferable to include a stabilizing amount of sodium sulfite or other antioxidant in the formulation.
  • a stabilizing amount of sodium sulfite or other antioxidant in the formulation.
  • the sodium sulfite is present in amounts in excess of that which saturates the solution.
  • the concentration of the chromagen mixture in the solution will usually be in the range of 0.5 to 10% by weight.
  • the application to the fecal specimen of a developer/complete reagent having a solvent system based on a solvent for iron protoporphyrins may be. used in a variety of FOBT formats.
  • the solution When used, as a developer., the solution will contain the hydroperoxide and be applied to the specimen on a leuco dye-impregnated matrix.
  • the solution When used in the form of a complete reagent, the solution will contain a leuco dye.
  • the multi-chromagen of the invention together with the hydroperoxide and, optionally, other additives such as those described above.
  • test matrices used in the FOBT methods of the invention may be made from a variety of porous materials such as cellulosics (wood, paper), ceramic, glass fibers, natural or synthetic cloth fibers, felt, and sponge.
  • Bibulous filter paper is commonly used and is preferred.
  • the present invention is directed in particular to those liquids that are useful as solvents for iron protoporphyrins. These liquids should preferably have an intermediate to high capacity to dissolve iron protoporphyrins at a pH in the range of about 6.5 to 7.5. In the present instance such capacity was determined by mixing 25 mg crystalline hemin with 1 ml of solvent at ambient temperature, and measuring the amount of hemin remaining undissolved. Liquids evaluated by this procedure that exhibited a high capacity for dissolving hemin included aprotic amides, sulfoxides, sulfones, pyridine and mixtures of certain amines and other organic solvents.
  • the amides that were found to be acceptable iron protoporphyrin solvents are of the formula
  • R1, R2. and R3 are the same or different and represent hydrogen, lower alkyl. phenyl or benzyl.. with the provisos that both of R 1 and R2 are not hydrogen and R 3 may be li.nked with R1 or R2 to form a 5- or 6-membered heterocycle.
  • the term "lower” as used to modify "alkyl” denotes moieties of 1 to 6 carbon atoms. Examples of such moieties are methyl. ethyl, isopropyl, butyl, and hexyl. Dimethyl formamide and l-methyl-2-pyrrolidinone are preferred solvents of this class.
  • R and R are the same or different and are lower alkyl, phenyl. benzyl, or. when X is
  • 1 2 R and R may be linked to form a 5 or 6 membered heterocycle (e.g.. tetramethylene sulfoxide. pentamethylene sulfoxide) .
  • a 5 or 6 membered heterocycle e.g.. tetramethylene sulfoxide. pentamethylene sulfoxide
  • the mixtures of neutralized amines and organic solvents that were found to be acceptable iron protoporphyrin solvents include: ethanolamine with glycerol, tetrahydrofurfuryl alcohol, 2-methoxy ethanol, methyl ethyl ketone. tetra ethyl urea, sulfolane (tetramethylene sulfone). or butyrolactone; 2-(diethylamino)ethylamine with methyl ethyl ketone, acetonitrile, sulfolane. butryolactone. tetrafurfuryl alcohol. 2-methoxy ethanol, or methanol and diethanolamine with methyl ethyl ketone.
  • iron protoporphyrin solvent is also preferably one that wets the test matrix so that it is capable of transporting (chromatographing) dissolved iron protoporphyrin away from the specimen so that the color change, if any. is more easily seen and not obscured by the specimen.
  • Co-solvents such as water, alkanols (e.g., methanol. ethanol, and other lower alkanols), pentane, ethyl acetate, cyclohexane. and acetone may be included with the iron protoporphyrin solvent, but the proportion of principal solvent in the solvent compositions should be maintained above about 50% (v/v) . preferably above about 75% (v/v) .
  • DMSO Dimethylsulfoxide
  • DMSO Dimethylsulfoxide
  • the use of DMSO as the solvent in the developer/complete reagent provides several advantages. Firstly, it is an excellent solvent for hemoproteins. including iron protoporphyrins. Secondly, it converts heme and hemin dimers and aggregates into the monomeric forms. And. thirdly, it inactivates peroxidases that may be present in the fecal specimen and inhibits them from catalyzing the oxidation of the indicator and producing a false positive result. Hei ⁇ oprotein solubiiizing agents that may be optionally included in tee solution or- test matrix include detergents and water-soluble polymers.
  • detergents that have suitable hydrophilic-hydrophobic balance to solubilize-hemoproteins are suitable.
  • Such detergents include the TRITON® detergents
  • the water-soluble polymers that may be used to solubilize hemoproteins include poly(eth ⁇ lene oxide), poly(vinyl alcohol). poly(vinyl pyrrolidone) . poly(vinyl pyridine). and poly(styrene sulfonate) . These solubiiizing agents not only solubilize hemoproteins. but are believed to convert peroxidatively inactive hemoprotein dimers or aggregates into peroxidatively active monomeric species.
  • Nitrogenous ligands that stabilize iron protoporphyrins and enhance their peroxidative activity may also be incorporated in the complete reagent or test matrix.
  • ligands are pyridine. histidine, caffeine, imidazole, and imidazole derivatives.
  • Quinoline and quinoline derivatives may be included in the developer/complete reagent to accelerate the color formation.
  • a preferred quinoline derivative is 7-chloro-4-(4-dieth ⁇ lamino-.l-methyl(butylamino)quino ⁇ line.
  • additives such as vegetable peroxidase inhibitors and iron chelators (e.g.. ethylenediamine tetraacetic acid N. N. N' . N' - diamino cyclohexane tetraacetic acid, citric acid, tartaric acid, nitrilotriacetic acid, diethylenetriamine-pentaacetic acid, N.N'-bishydroxyethyl glycine, ethyleneglycol bis(2-aminoethylether)tetraacetic acid.
  • N-hydroxyethyl- ethylenediaminetetraacetic acid may be incorporated into the test matrix to further reduce the likelihood of false test results.
  • Buffers are added to maintain a suitable pH range for oxidizing the leuco dye.
  • the particular buffer pH range will depend on the leuco dye that is used.
  • the pH will usually be between about 3 and about 9.
  • guaiac oxidation is buffered at pH 6-7.5 (phosphate buffer).
  • 3.3' .5.5'-tetramethylbenzidine oxidation is buffered at a pH of about 4 (acetate buffer)
  • ABTS is buffered at a pH of about 9-9.5 (glycine buffer).
  • the pH should be in the range of 5 to 10.
  • Hydrogen peroxide or organic hydroperoxides such as cu ene hydroperoxide. t-butyl hydroperoxide, diisopropylbenzene hydroperoxide, and 2,5-dimethylhexane hydroperoxide may be used in the developer or complete reagent. It is preferred to use an organic hydroperoxide since organic hydroperoxides are less likely to (a) produce false positive results in FOBT in which vegetable peroxidases are present in the fecal specimen and (b) destroy the peroxidase activity of the hemoprotein.
  • the concentration of hydroperoxide in the developer/complete reagent will usually be in the range of 0.05- to 10% by volume, more usually 0.5 to 5% by volume.
  • Example 1 Tests were carried out to compare the specificity and sensitivity achieved in FOBT with a developer composition consisting of (5% (v/v) cumene hydroperoxide (CHP) in DMSO) versus that achieved with a conventional FOBT developer (3-6% (v/v) hydrogen peroxide in ethanol/water) .
  • a developer composition consisting of (5% (v/v) cumene hydroperoxide (CHP) in DMSO) versus that achieved with a conventional FOBT developer (3-6% (v/v) hydrogen peroxide in ethanol/water) .
  • the DMSO-based developer was more sensitive than the conventional developer with hemoglobin and hemin and much less sensitive than the conventional developer with horseradish peroxidase.
  • the invention developer thus improves the sensitivity and specificity of the test. Sensitivity can be improved by modifying the developer as needed.
  • Hemin was dissolved in a 1:10 (v/v) mixture of DMSO and water. Two u-1 of this solution was spotted onto filter paper and the paper was air dried. Spotted areas were then treated with 5 u.1 of one of the following developers: (a) CHP, 5%. in DMSO; (b) t-butyl hydroperoxide. 5%. in DMSO; (c) H O . 5% in water; (d) HEMOCCULT® developer (H O 5%, in ethanol/ water); (e) DMSO alone; (f) water alone;, and (g) 75% ethanol. 25% water.
  • developers (a) CHP, 5%. in DMSO; (b) t-butyl hydroperoxide. 5%. in DMSO; (c) H O . 5% in water; (d) HEMOCCULT® developer (H O 5%, in ethanol/ water); (e) DMSO alone; (f) water alone;, and (g) 75%
  • Tests were conducted using developer compositions consisting of CHP at 5% in DMSO with various cosolvents. The procedure of Example 1 was used except that the highest clearly detectable dilution of catalyst is noted rather than color intensity.
  • Tests were carried out to assess the effect of the developer composition on the mobility of hemin spotted on filter paper. This is an indirect measure of the solubility of hemin in the developer.
  • Whatman filter paper was soaked with 10 ⁇ l of a 1 mg/ml hemin solution in 1 mM aqueous ammonium hydroxide. After drying, a small section of the hemin-treated paper was placed- on a HEMOCCULT® slide. Ten ⁇ l of developer compositions comprising 5% CHP in DMSO/water solvent containing 100%, 95%. 90%. 80%. 75%, 50%. and 25% (v/v) DMSO were placed on the hemin spots and blue color " formation was observed. At DMSO concentrations above about 75% blue color was noted in the entire slide area wetted by the developer. At lower DMSO concentrations color was observed only directly under the hemin-treated filter paper. A similar test with HEMOCCULT® developer produced no color.
  • Example 5 This example illustrates the effect of .hemin solubility on the readability of FOBT test ⁇ esults.
  • a second FOBT formulation was prepared in tetramethyl urea (2% guaiac, 1% ABTS and 5% cumene hydroperoxide) .
  • Fifty mi ⁇ roliters of the new FOBT formulation in tetramethyl urea was added to a smear of the spiked fecal specimen on filter paper. No blue color was eluted from the specimen onto the surrounding filter paper. The specimen became somewhat darker in appearance. but it was extremely difficult to decide if a blue color was present because of the dark fecal background.
  • DMSO is a good heme solvent, and a 1:1 mixture of tetramethyl urea and aqueous 1 M ethanolamine. pH 7. is a good solvent for hemin even though ' neither tetramethyl urea nor aqueous 1 M ethanolamine alone is a good hemin solvent.
  • Hemoglobin was denatured by treatment with (a) hydrochloric acid, (b) enzymes or (c) heat as follows: (a) Acid
  • Dilute (0.1 M) hydrochloric acid was added to a solution of hemoglobin in water (5 mg/ml) until the pH of the solution was 1.0- The solution was incubated for 1 hr at 37°C and the pH adjusted to 7.4 with 0..1 N sodium hydroxide. The resulting material was homogenized to produce a finely dispersed suspension and incubated at 37°C for 48 hr.
  • This example illustrates the enhanced readability of the multi-chromagen FOBT reagent as regards color intensity and diffusiveness.
  • the following reagents were prepared:
  • HEMOCCULT® developer (6% hydrogen peroxide in alcohol):
  • Standard HEMOCCULT® slides were smeared with untreated fecal specimens, or with specimens which had been spiked with hemin or nemoglobin (135 or 270 ⁇ g hemin/g feces. or with 5.7 mg hemoglobin/g feces). After the smears had dried, the appropriate reagent was added, and the appearance of the test noted at 1 min. The results are reported in Table 4.
  • This example shows the relative sensitivity and readability of FOBTs using the multi-chromagen of the invention and reagents containing only ABTS or guaiac.
  • Reagent A Blue ring at solvent front, but hollow. colorless center.
  • Reagent B No color formed on standard filter pape .
  • Reagent C Very intense blue color formed; color evenly distributed throughout the entire solvent area.
  • This example shows the chromatographic advantages of the multi-chromagen reagent relative to a single chromagen reagent.
  • Reagent B Multi-chromagen reagent (2% guaiac, 5% cumene hydroperoxide, and 25 mg/ml ABTS in DMSO over 100 mg solid sodium sul ite),
  • This example illustrates enhanced color intensity and uniformity obtained using the multi-chromagen reagent.
  • the following reagents were used:
  • Intensity Appearance Intensity Appearance 1. +3 Blue ring with +10 Solid blue circle hollow center intensely blue
  • Blue ring with hollow center Blue color at edge of solvent front, but no color in center.
  • Solid blue circle Even, blue color throughout the area covered by the solvent.
  • This example provides a comparison of color intensity, stability and chromatography in tests using the HEMOCCULT® reagents and the multiple-chromagen of the invention, with- hemin as catalyst.
  • HEMOCCULT® developer (6% hydrogen peroxide in alcohol) .
  • Multi-chromagen reagent (1% guaiac, 5% cumene hydroperoxide. and ABTS (25 mg/ml) in DMSO).
  • Reagent A Weak, diffuse color with streaking.
  • Reagent B Extremely intense blue color. Color is evenly distributed throughout the area covered by the solvent.
  • This example reports the sensitivity of complete multiple-chromagen FOBT to plant peroxidases relative to- other reagents.
  • the following reagents were used:
  • HEMOCCULT® developer (6% hydrogen peroxide in alcohol).
  • This example shows the effect of sulfite on the multiple-chromagen reagent stability.

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EP19870903812 1986-06-02 1987-06-01 Testreagenzien und verfaren zur bestimmung von okkultem blut in fäkalien. Ceased EP0267952A4 (de)

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US86957386A 1986-06-02 1986-06-02
US869573 1986-06-02

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US4939097A (en) * 1986-06-02 1990-07-03 Litmus Concepts, Inc. Fecal occult blood test methods
US4971914A (en) * 1984-12-11 1990-11-20 Litmus Concepts, Inc. Developer for fecal occult blood tests
WO1990002339A1 (en) * 1988-08-16 1990-03-08 Cetus Corporation Stable indicator solutions for detection of peroxidatic activity
WO1990002202A1 (en) * 1988-08-16 1990-03-08 Cetus Corporation Reduction of peroxidatic and catalatic interference with assays of peroxidatic activity
JP2660558B2 (ja) * 1988-08-17 1997-10-08 大日本印刷株式会社 精製グアヤク脂およびその製造法
US5025504A (en) * 1988-12-16 1991-06-25 Weyerhaeuser Company Liner for a helmet, hat, cap or other head covering
US5176999A (en) * 1989-12-07 1993-01-05 Eastman Kodak Company Buffered wash composition, insolubilizing composition, test kits and method of use
AU9438498A (en) * 1997-08-28 1999-03-22 Societe Des Produits Nestle S.A. Test method for peroxidase
CN114965618B (zh) * 2022-06-13 2023-10-17 潍坊学院 双模式生物传感器及其在dna甲基转移酶活性检测中的应用
CN118275668B (zh) * 2024-04-10 2024-11-19 上海京都生物工程有限公司 一种大便隐血测试卡的制备及其使用方法

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See also references of WO8707718A1 *

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EP0267952A1 (de) 1988-05-25
WO1987007718A1 (en) 1987-12-17
JPH087208B2 (ja) 1996-01-29
JPS63503483A (ja) 1988-12-15

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