ANTIBODY DETECTION

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: 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.

Inventors: STARKIE SELBY JOHN, WILLIAMS GWYNFOR REES, STARKIE, SELBY, JOHN, WILLIAMS, GWYNFOR, REES
Applicants: AXON HEALTHCARE LTD, AXON HEALTHCARE LTD.
Application year: 1985
Main classification: G01N33
IPC codes: G01N33