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)

Applications Claiming Priority (2)

Publications (2)

Family

ID=23149085

Family Applications (1)

Country Status (5)

Families Citing this family (10)

Citations (1)

Family Cites Families (5)

• 1982
  • 1982-08-12 WO PCT/US1982/001107 patent/WO1983000877A1/en not_active Application Discontinuation
  • 1982-08-12 EP EP19820902930 patent/EP0091911A4/de not_active Withdrawn
  • 1982-08-30 ES ES515350A patent/ES515350A0/es active Granted
  • 1982-08-30 CA CA000410409A patent/CA1206092A/en not_active Expired
  • 1982-08-31 IT IT23075/82A patent/IT1153188B/it active
• 1983
  • 1983-07-16 ES ES524183A patent/ES524183A0/es active Granted

Patent Citations (1)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events