WO1986000994A1 - Antibody detection - Google Patents

Antibody detection Download PDF

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Publication number
WO1986000994A1
WO1986000994A1 PCT/GB1985/000342 GB8500342W WO8600994A1 WO 1986000994 A1 WO1986000994 A1 WO 1986000994A1 GB 8500342 W GB8500342 W GB 8500342W WO 8600994 A1 WO8600994 A1 WO 8600994A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
specimen
monoclonal antibody
detection
bound
Prior art date
Application number
PCT/GB1985/000342
Other languages
French (fr)
Inventor
Selby John Starkie
Gwynfor Rees Williams
Original Assignee
Axon Healthcare Ltd.
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 Axon Healthcare Ltd. filed Critical Axon Healthcare Ltd.
Publication of WO1986000994A1 publication Critical patent/WO1986000994A1/en

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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57449—Specifically defined cancers of ovaries
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • a method for antibody detection according to the present invention, comprises
  • part at least, and usually most, of the bound antibody is removed in order to make antigen available for reaction with monoclonal antibody.
  • the specimen is taken from, for example, cells suspected of containing antigen-bound antibody, e.g. a tumour.
  • the specimen may be isolated, e.g. conveniently in the form of a tissue section on a slide.
  • Part of the antibody is then removed, e.g. by elution, and remaining, bound antibody is blocked.
  • blocking may be achieved by treatment with goat antiserum against human immunoglobulin.
  • the monoclonal antibody which is used in the second step of the method of the invention is chosen to be specific with respect to the antigen which binds the antibody to be detected.
  • the method can be adapted for detection of more than one antibody, by the use of various monoclonal antibodies at this stage of the ethod. In any case, it may be desirable to use various monoclonal antibodies, as controls.
  • the specimen contains antigen-bound monoclonal antibody.
  • the monoclonal antibody is marked, e.g. with a fluorescent or other label.
  • a label may be introduced by reacting with the human monoclonal antibody with goat antiserum against human immunoglobulin conjugated with peroxidase. Detection may then be conducted in conventional manner.
  • the method of the invention allows simple detection of, for example, antibody against tumours from the same human or other subject. It can be used to detect antibodies in cells isolated from human tissue. In particular, it can be used for the detection of antibody activity produced by the procedure described in an International Patent Application entitled "Antibody Production” filed in the names of Axon Healthcare Ltd., S. J. Starkie and G. R. Williams on 30th July 1985. The following Examples illustrate the invention.
  • Example 1 illustrates the invention.
  • Human B lymphocytes isolated from the excised oligodendroglioma of a patient were cultured in growth medium. Parts of the excised oligodendroglioma were frozen to the temperature of liquid nitrogen; 5 ⁇ m sections were cut with a cryostat and mounted on glass slides. The patient's antibody was removed from the sections by immersion in citrate-buffered saline (pH 3.5) for 60 minutes. The sections were dried with acetone for 10 minutes. Remaining antibody was blocked by immersing the sections in a solution of goat antiserum against human immunoglobulin (0.035 g/ml) for 30 minutes.
  • the sections were washed with tris-saline (pH 7.6) and immersed in the specific monoclonal antibody for 60 minutes; control sections were immersed in other monoclonal antibodies and in a saline solution (0.84 g/1) . They were washed with tris-saline and immersed in a 1/50 solution of goat antiserum against human immunoglobulin (conjugated with peroxidase) for 30 minutes. The sections were washed with tris-saline and immersed in a solution of diaminobenzidine for 10 minutes. They were washed in distilled water and immersed in Harris haematoxylin for 2 minutes.
  • Example 2 The procedure of Example 1 was repeated, but using lymphocytes isolated from a human ovarian carcinoma.
  • Example 3 The procedure of Example 1 was repeated, but using lymphocytes isolated from a human ovarian carcinoma.
  • Example 4 The procedure of Example 1 was repeated, but using lymphocytes isolated from human breast cancer tissue.
  • Example 4 The procedure of Example 1 was repeated, but using lymphocytes isolated from human breast cancer tissue.
  • Example 5 The procedure of Example 1 was repeated, but using lymphocytes isolated from a human colon carcinoma.
  • the "other" tissues are each of oligodendroglioma from another subject, cerebrum, cerebellum, mid-brain, ovarian carcinoma from two subjects, large bowel, small bowel, stomach, testis, spleen, thyroid and early placenta.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

A method for antibody detection, which comprises (1) treating a specimen containing antigen-bound antibody, in order to remove some of the antibody, and blocking remaining, bound antibody; (2) exposing the treated specimen to monoclonal antibody; (3) marking the monoclonal antibody-carrying specimen with a marker; and (4) detecting the marker.

Description

ANTIBODY DETECTION FIELD OF THE INVENTION
This invention relates to antibody detection. STATEMENT OF THE INVENTION A method for antibody detection, according to the present invention, comprises
(1) treating a specimen containing antigen-bound antibody, in order to remove some of the antibody, and m blocking remaining, bound antibody; (2) exposing the treated specimen to monoclonal antibody;
(3) marking the monoclonal antibody-carrying specimen with a marker; and
(4) detecting the marker. DETAILED DESCRIPTION OF THE INVENTION
In the first step of the method of the invention, part at least, and usually most, of the bound antibody is removed in order to make antigen available for reaction with monoclonal antibody. The specimen is taken from, for example, cells suspected of containing antigen-bound antibody, e.g. a tumour. The specimen may be isolated, e.g. conveniently in the form of a tissue section on a slide. Part of the antibody is then removed, e.g. by elution, and remaining, bound antibody is blocked. For example, if the subject is a human whose specific antibody has been isolated for use in the detection method, blocking may be achieved by treatment with goat antiserum against human immunoglobulin.
The monoclonal antibody which is used in the second step of the method of the invention is chosen to be specific with respect to the antigen which binds the antibody to be detected. The method can be adapted for detection of more than one antibody, by the use of various monoclonal antibodies at this stage of the ethod. In any case, it may be desirable to use various monoclonal antibodies, as controls.
At this stage, the specimen contains antigen-bound monoclonal antibody. In the third step of the method, the monoclonal antibody is marked, e.g. with a fluorescent or other label. For example, a label may be introduced by reacting with the human monoclonal antibody with goat antiserum against human immunoglobulin conjugated with peroxidase. Detection may then be conducted in conventional manner.
The method of the invention allows simple detection of, for example, antibody against tumours from the same human or other subject. It can be used to detect antibodies in cells isolated from human tissue. In particular, it can be used for the detection of antibody activity produced by the procedure described in an International Patent Application entitled "Antibody Production" filed in the names of Axon Healthcare Ltd., S. J. Starkie and G. R. Williams on 30th July 1985. The following Examples illustrate the invention. Example 1
Human B lymphocytes isolated from the excised oligodendroglioma of a patient were cultured in growth medium. Parts of the excised oligodendroglioma were frozen to the temperature of liquid nitrogen; 5 μm sections were cut with a cryostat and mounted on glass slides. The patient's antibody was removed from the sections by immersion in citrate-buffered saline (pH 3.5) for 60 minutes. The sections were dried with acetone for 10 minutes. Remaining antibody was blocked by immersing the sections in a solution of goat antiserum against human immunoglobulin (0.035 g/ml) for 30 minutes.
The sections were washed with tris-saline (pH 7.6) and immersed in the specific monoclonal antibody for 60 minutes; control sections were immersed in other monoclonal antibodies and in a saline solution (0.84 g/1) . They were washed with tris-saline and immersed in a 1/50 solution of goat antiserum against human immunoglobulin (conjugated with peroxidase) for 30 minutes. The sections were washed with tris-saline and immersed in a solution of diaminobenzidine for 10 minutes. They were washed in distilled water and immersed in Harris haematoxylin for 2 minutes. They were then washed in running tap water for 15 minutes, dried with absolute methanol and then xylol, and mounted with DePeX under a glass cover slip. The binding of the monoclonal antibody was indicated by a brown precipitate on the section when inspected by light microscopy. Example 2 The procedure of Example 1 was repeated, but using lymphocytes isolated from a human ovarian carcinoma. Example 3
The procedure of Example 1 was repeated, but using lymphocytes isolated from human breast cancer tissue. Example 4
The procedure of Example 1 was repeated, but using lymphocytes isolated from a human colon carcinoma. Example 5
Parts of normal tissue excised from patients and from cadavers were frozen to the temperature of liquid nitrogen. They were cut into sections and treated in the same way, with human monoclonal antibodies derived from cancer patients, as in previous Examples.
Many of the normal tissues were coated with human immunoglobulin before elution, but the immunoglobulin was removed by elution at pH 3.5. The binding of human monoclonal antibody with normal tissue indicated the binding specificity of the antibody. The binding pattern of ten antibodies from an oligodendroglioma patient (DS) in an immunoperoxidase assay is shown in the following Table. The values in the Table indicate the observed degree of binding on a scale of 0 (no binding) up to 4
(nt means not tested) .
The "other" tissues are each of oligodendroglioma from another subject, cerebrum, cerebellum, mid-brain, ovarian carcinoma from two subjects, large bowel, small bowel, stomach, testis, spleen, thyroid and early placenta.
Table Antibody DS Tissue Tonsil Tissue Other Tissues
DS1A Nl 1 1 0
DS1A N7 3 0 0
DS1A N14 4 0 0
DS1A N15 2 1 0 DS1A NA 2 0 0
DS1A NB 1 0 0
DS1A NC 2 0 0
DS1A FA 2 0 0
DS1A FB 1 0 0 DS1A NO 2 nt 0

Claims

1. A method for antibody detection, which comprises
(1) treating a specimen containing antigen-bound antibody, in order to remove some of the antibody, and blocking remaining, bound antibody;
(2) exposing the treated specimen to monoclonal antibody;
(3) marking the monoclonal antibody-carrying specimen with a marker; and (4) detecting the marker.
2. A method according to claim 1, in which the specimen is derived from tumour tissue.
3. A method according to claim 1 or claim 2, in which the monoclonal antibody is specific with respect to the antigen which binds the antibody to be detected.
PCT/GB1985/000342 1984-07-31 1985-07-30 Antibody detection WO1986000994A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB848419458A GB8419458D0 (en) 1984-07-31 1984-07-31 Antibody detection
GB8419458 1984-07-31

Publications (1)

Publication Number Publication Date
WO1986000994A1 true WO1986000994A1 (en) 1986-02-13

Family

ID=10564694

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1985/000342 WO1986000994A1 (en) 1984-07-31 1985-07-30 Antibody detection

Country Status (4)

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EP (1) EP0190245A1 (en)
JP (1) JPS61502840A (en)
GB (1) GB8419458D0 (en)
WO (1) WO1986000994A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330902A2 (en) * 1988-02-24 1989-09-06 BEHRINGWERKE Aktiengesellschaft Use of monoclonal antibodies in the performance control of immunometric assays

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2098730A (en) * 1981-04-21 1982-11-24 Welsh Nat School Med Immunolocalisation
EP0080109A1 (en) * 1981-11-19 1983-06-01 New York Blood Center, Inc. Sensitive immunoassays of antigens or antibodies sequestered with immune complexes
EP0087898A1 (en) * 1982-02-22 1983-09-07 Cancer Research Campaign Technology Limited Antibodies and antigens useful in the diagnosis and treatment of cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2098730A (en) * 1981-04-21 1982-11-24 Welsh Nat School Med Immunolocalisation
EP0080109A1 (en) * 1981-11-19 1983-06-01 New York Blood Center, Inc. Sensitive immunoassays of antigens or antibodies sequestered with immune complexes
EP0087898A1 (en) * 1982-02-22 1983-09-07 Cancer Research Campaign Technology Limited Antibodies and antigens useful in the diagnosis and treatment of cancer

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0330902A2 (en) * 1988-02-24 1989-09-06 BEHRINGWERKE Aktiengesellschaft Use of monoclonal antibodies in the performance control of immunometric assays
EP0330902A3 (en) * 1988-02-24 1990-10-24 BEHRINGWERKE Aktiengesellschaft Use of monoclonal antibodies in the performance control of immunometric assays

Also Published As

Publication number Publication date
EP0190245A1 (en) 1986-08-13
GB8419458D0 (en) 1984-09-05
JPS61502840A (en) 1986-12-04

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