EP0091911A1 - Detection immunohistochimique a l'oxydase de glucose d'anticorps anti-nucleaires - Google Patents

Detection immunohistochimique a l'oxydase de glucose d'anticorps anti-nucleaires

Info

Publication number
EP0091911A1
EP0091911A1 EP82902930A EP82902930A EP0091911A1 EP 0091911 A1 EP0091911 A1 EP 0091911A1 EP 82902930 A EP82902930 A EP 82902930A EP 82902930 A EP82902930 A EP 82902930A EP 0091911 A1 EP0091911 A1 EP 0091911A1
Authority
EP
European Patent Office
Prior art keywords
antigen source
source
contacting
sheath
support
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP82902930A
Other languages
German (de)
English (en)
Other versions
EP0091911A4 (fr
Inventor
Tara Rathlev
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ICL SCIENTIFIC
Original Assignee
ICL SCIENTIFIC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ICL SCIENTIFIC filed Critical ICL SCIENTIFIC
Publication of EP0091911A1 publication Critical patent/EP0091911A1/fr
Publication of EP0091911A4 publication Critical patent/EP0091911A4/fr
Withdrawn legal-status Critical Current

Links

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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G01N33/567—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds utilising isolate of tissue or organ as binding agent
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica

Definitions

  • This invention relates generally to immuno ⁇ enzymatic processes for use in medical diagnoses, and more particularly, to an immunohistochemical process for the detection of antinuclear antibodies in test serum.
  • an increasing amount of research has focused on improving clinical laboratory tests.
  • a primary thrust of this research has been to replace the traditional radiotracer labels with safer and more stable labels.
  • fluorescent markers have found wide acceptance. While fluorescent markers give quick results, they have not proven entirely satis- factory for a number of assays because once placed in a detectable state they have a very limited shelf life, fading after a few days or more. Moreover, fluorescent assays frequently exhibit high background due to auto-
  • horseradish peroxidase has not proven entirely satis ⁇ factory, however, because of a high tendency to produce non-specific background stain, even in spite of attempts to quench the presence of endogenous peroxidase-like activity.
  • Another objectionable feature of the peroxi ⁇ dase label is that it generally requires the use of a highly carcinogenic material, diaminobenzidine, for superior stain development. Although substitutes for this material have been developed, their contrast and intensity are typically not entirely satisfactory.
  • the present invention provides an immunohisto- chemical method which substantially reduces the amount of background stain caused by endogenous materials, while producing a relatively permanent record of the assay. Moreover, the reagents of the present invention are relatively inexpensive to manufacture, are stable for long periods of time even when stored at room tempera ⁇ ture, and have minimal carcinogenic characteristics.
  • an immuno ⁇ enzymatic process for the detection of a component of a test sample comprising steps of: contacting the test sample with an antigen source fixed onto support and stabilized by dehydration; contacting the antigen source with an antibody conjugated with glucose oxidase and reactive with the component; incubating the antigen source with the solution containing glucose and a chro ogenic mixture and; analyzing the antigen source for the presence of color from the incubation.
  • the chromogenic mixtures preferably contain t-nitroblue tetrazolium chloride and m-phenazine metho- sulfate, which permit analysis with a light microscope.
  • the component of the test sample is an antinuclear antibody
  • the antigen source fixed on a glass slide is preferably Hep-2 cells or rat kidney tissue, and the conjugated antibody is goat immunoglobulin reactive with human immunoglobulins.
  • an immunohistoche ical method for detecting antinuclear antibodies in a test serum, substantially in the absence of background stain ⁇ ing comprises steps of: providing a nuclear antigen source fixed onto a slide and dehydrated with acetone at about 4 * C;
  • u ⁇ *- U contacting the nuclear antigen source with the test serum; contacting the nuclear antigen source with goat immunoglobulin conjugated with glucose oxidase and reactive with the .antinuclear antibodies; incubating the nuclear antigen source with a solution containing beta-D-glucose, t-nitroblue tetra- zolium, and 1-methoxyphenazine methosulfate; and analyzing the nuclear antigen source under a light microscope for the presence of a formazan stain.
  • Another aspect of the invention is a method of stabilizing an im unochemical substrate fixed on a solid support, the method comprising the steps of: providing a substrate fixed on the support; contacting the substrate with an organic solvent reagent to substantially dehydrate the substrate; placing the support into a protective sheath; backfilling the sheath with a gas; and sealing the support in the sheath.
  • the support is preferably a glass slide, and the substrate Hep-2 cells or rat kidney tissue.
  • the organic solvent reagent can be composed of acetone, methanol, or mixtures thereof, and the interacting step performed between about 4 and 8° C.
  • the sheath can be composed of a polyester film, and can contain a des- sicant.
  • the preferred gas is nitrogen gas.
  • Exemplary starting materials useful in practicing the present invention are slides fixed with human epethelial cells (Hep-2) or with rat kidney tissue suitable for use in antinuclear antibody assays.
  • Such slides can be purchased commercially from a number of sources, including Antibodies Incorporated, Davis, California, and Behring, La Jolla, California, or, alternatively, can be manufactured according to con- ventional techniques. Generally, these slides should be stored at -20°C. to prevent degradation.
  • the slides are immersed ' for about 10 minutes in a bath containing histological grade acetone, methanol, or a combination of these organic solvents.
  • the bath is maintained at a temperature preferably ranging from about 4 C. to about 8 C.
  • After removing the slides from the bath they are allowed to air dry for about 2 to 3 minutes.
  • the slides are preferably stored at about 5°C. during continued processing.
  • the slides are placed into a protective sheath, preferably made from a poly ⁇ ethylene film, such as MYLAR bags with an aluminum outer layer.
  • a protective sheath preferably made from a poly ⁇ ethylene film, such as MYLAR bags with an aluminum outer layer.
  • a small packet containing a standard dessicant is placed in the bag, and the bag is partially sealed. Nitrogen gas is then introduced through the unsealed portion of the bag until the bag bulges slightly. The bag is then totally sealed to prevent the nitrogen gas from escaping.
  • O PI Goat immunoglobulin conjugated with the glucose oxidase enzyme can be prepared as follows. Glucose oxidase, which is available from a variety of commercial sources, including Sigma, St. Louis, Missouri, and Boehringer Manneheim, Indianapolis, Indiana, is dissolved in distilled water at a concentration of about 12 mg/ml. To this solution is added about 0.2 ml of ,0.1 M sodium periodate. The mixture is stirred for about 20 minutes and then dialyzed, preferably against 1 mM sodium acetate buffer, pH 4.4, for about 16 hours.
  • the glucose oxidase solution is mixed with 1 ml of 0.01 M sodium bicarbonate buffer, pH 9.5, containing about 8 mg of the IgG fraction of goat anti- human im unoglobulins (IgG, IgA, and IgM) .
  • 0.1 mis of. an aqueous solution containing about 4 mg/ml sodium borohydrate water solu ⁇ tion is added.
  • the entire mixture can be chromatographed on a gel filtration column, such as a Sephacryl S-300, available from Pharmacia, Piscataway, New Jersey, and the fractions containing the enzyme-IgG conjugate pooled. This pool can then be titrated. to determine working dilutions.
  • a method for detecting antinuclear antibodies utilizing these reagents is performed as follows.
  • a small amount of test serum, or other test solution, is contacted with the nuclear antigen fixed source on the slide. After about thirty minutes at room temperature, the serum is washed from the slide with phosphate buf ⁇ fered saline (PBS) , and then soaked in a cold PBS solu ⁇ tion for about 10 minutes. The slides are removed from the PBS and excess moisture blotted with absorbent paper.
  • a small amount of the enzy e-IgG conjugate, preferably about 50 ul, is contacted with the antigen source for about 30 minutes at room temperature. The slide is again washed with PBS, soaked for 10 minutes in a cold PBS solution, removed, and excess moisture blotted with absorbent paper.
  • the color reagent may be pre ⁇ pared. It consists of about 2 volumes beta-D-glucose, 15 mg/ml in 0.1 M sodium phosphate buffer, pH 6.9, one volume t-nitroblue tatrazolium (t-NBT) , 2 mg/ml in the same buffer (if necessary, this may be filtered through Whattman No. 1 filter paper just prior to use) , and 1 volume of 1-methoxyphenazine methosulfate (m-PMS), 0.8 mg/ml in the same buffer.
  • t-NBT t-nitroblue tatrazolium
  • m-PMS 1-methoxyphenazine methosulfate
  • the preferred chro o- genic reagents are t-NBT and m-PMS, other materials can also be utilized.
  • the mixture may be prepared about 1 hour before the staining reaction.
  • a small volume, preferably about 100 ul, of the color reagents are contacted wtih the antigen source.
  • the slides are placed in a slide chamber and incubated for about 30 minutes at 55 °C. to permit formation of the formazan stain.
  • the excess color reagent is then washed from the slide with PBS, soaked for about 15 minutes in cold PBS and the excess moisture again removed with the absorbent paper.
  • the slide is then prepared for light microsoope viewing according to standard techniques. Briefly, a glycerol-gelatin mounting medium is placed in a 55°C. incubator for about 5 to 10 minutes for liquefication, and a large drop placed over the antigen source. The antigen source is then covered with a cover slip, taking care to avoid formation of air bubbles. When the slides are examined under a light microscope, the existence of antinuclear antibodies in the test serum will cause slightly enlarged, homo ⁇ geneous dark nuclei to appear, substantially without dark background staining.
  • the present invention provides an ef ⁇ fective, inexpensive, highly visible, and permanent record of an antinuclear antibody assay. Further, the nuclear antigen source fixed onto the slide is extremely stable and does not require storage at low temperatures.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Inorganic Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

On utilise, pour la détection immunohistochimique d'antigènes et d'anticorps apparentés dans un sérum de test, une source antigène fixée sur un support et stabilisée par un traitement de déshydratation avec un mélange de solvant organique, tel que du méthanol d'acétone, ou des mélanges de celui-ci. Le support est stocké sous une atmosphère d'azote dans un sachet de polyéthylène scellé. En outre, un procédé immunoenzymatique de détection des composants biologiques pour un échantillon d'analyse de test, le procédé comprenant les étapes suivantes: la source antigène stabilisée est mise en contact avec l'échantillon de test, la source antigène est mise en contact avec un anticorps conjugué avec de l'oxydase de glucose et réagissant avec ce composant, incubation de la source antigène avec une solution contenant du glucose et un mélange chromagène, et analyse de la source antigène pour détecter la présence de couleur, de préférence avec un microscope lumineux.
EP19820902930 1981-08-31 1982-08-12 Detection immunohistochimique a l'oxydase de glucose d'anticorps anti-nucleaires. Withdrawn EP0091911A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US29811281A 1981-08-31 1981-08-31
US298112 1999-04-23

Publications (2)

Publication Number Publication Date
EP0091911A1 true EP0091911A1 (fr) 1983-10-26
EP0091911A4 EP0091911A4 (fr) 1984-04-06

Family

ID=23149085

Family Applications (1)

Application Number Title Priority Date Filing Date
EP19820902930 Withdrawn EP0091911A4 (fr) 1981-08-31 1982-08-12 Detection immunohistochimique a l'oxydase de glucose d'anticorps anti-nucleaires.

Country Status (5)

Country Link
EP (1) EP0091911A4 (fr)
CA (1) CA1206092A (fr)
ES (2) ES8400773A1 (fr)
IT (1) IT1153188B (fr)
WO (1) WO1983000877A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4487830A (en) * 1982-05-14 1984-12-11 American Hoechst Corporation Enzyme/immunofluorescent assay for autoantibodies
US4588682A (en) * 1982-12-13 1986-05-13 Integrated Genetics, Inc. Binding nucleic acid to a support
CA1279818C (fr) * 1985-03-29 1991-02-05 Cenfold Holdings S.A. Test pour la detection des anticorps contre certains micro-organismes
EP0206779A1 (fr) * 1985-06-21 1986-12-30 Modern Diagnostics, Inc. Détection des anticorps antinucléaires
AU7140287A (en) * 1986-04-08 1987-10-15 Meloy Laboratories, Inc. Immunofluorescent microscopy substrate fixation
CA1313818C (fr) * 1987-06-03 1993-02-23 Ross Leon Coppel Antigene nucleaire la
EP0335293A3 (fr) * 1988-03-30 1990-09-12 Hoechst Japan Limited Procédé pour préparer un élément analytique pour la détermination d'anticorps anti-nucléaire
US7902162B2 (en) 2004-02-20 2011-03-08 Beijin Xinjing Antai Medical and Technology Service Limited Corp Medicament and method for treating recurrent spontaneous abortion
CN100504388C (zh) * 2004-02-20 2009-06-24 北京新景安太医疗技术服务有限公司 免疫性反复自然流产的诊断方法和治疗监测方法
US7763418B2 (en) * 2005-07-05 2010-07-27 Cytoskeleton, Inc. Detection of Rho proteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0008720A1 (fr) * 1978-08-19 1980-03-19 BEHRINGWERKE Aktiengesellschaft Agent et méthode de diagnostic pour la détection d'anticorps mitochondriaires ou nucléaires

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3146163A (en) * 1962-01-23 1964-08-25 John H Brewer Apparatus for separating certain components from blood
NL154600B (nl) * 1971-02-10 1977-09-15 Organon Nv Werkwijze voor het aantonen en bepalen van specifiek bindende eiwitten en hun corresponderende bindbare stoffen.
US3997657A (en) * 1973-10-15 1976-12-14 American Cyanamid Company Dry slide reagent employed in immunofluorescent test for detection of human antinuclear factor
US3876504A (en) * 1974-06-10 1975-04-08 Early Warning Co Procedure for determination of antigens and antibodies and articles for use therewith
US3979509A (en) * 1974-09-03 1976-09-07 General Electric Company Opaque layer method for detecting biological particles

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0008720A1 (fr) * 1978-08-19 1980-03-19 BEHRINGWERKE Aktiengesellschaft Agent et méthode de diagnostic pour la détection d'anticorps mitochondriaires ou nucléaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO8300877A1 *

Also Published As

Publication number Publication date
EP0091911A4 (fr) 1984-04-06
ES515350A0 (es) 1983-11-01
ES8500999A1 (es) 1984-11-01
ES524183A0 (es) 1984-11-01
CA1206092A (fr) 1986-06-17
WO1983000877A1 (fr) 1983-03-17
IT1153188B (it) 1987-01-14
ES8400773A1 (es) 1983-11-01
IT8223075A0 (it) 1982-08-31

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Inventor name: RATHLEV, TARA