DD283303A7 - IMMUNOLOGICAL DETECTION PROCEDURE FOR SPECIFIC AUTOANTIKOERPER - Google Patents

Abstract

The invention relates to an immunological determination method according to which epitope- or antigen-specific autoantibodies in serum and other body fluids can be determined even in the presence of high immunoglobulin concentration, without the antigen ever having been isolated or its structure cleared up. According to the invention this is achieved by the use of monoclonal antibodies which are competitively inhibited in their specific antigen binding by serum antibodies of the same specificity, i. H. only if the antibody in question is present in the serum is the monoclonal antibody not bound or bound to a slight extent. In order to measure its displacement, either the monoclonal antibody itself is labeled or bound by a different labeled antibody as an antigen dependent concentration and thus detected. The field of application is clinical chemistry. {Autoantibodies, eptiospecific, antigen-specific; biological fluids; monoclonal antibodies; Mark; competitive inhibition; Verdraengungsmessung}

Description

Field of application of the invention

The invention relates to an immunological assay for the detection of diagnostically important autoantibodies predominantly in human, but also in animal blood serum and other body fluids.

Characteristic of the known state of the art

The detection of specific autoantibodies can so far only be done by using the isolated pure antigens. Either the antigen itself is labeled with an indicator substance, e.g. B. with an enzyme or radioactive isotope and its binding by antibody after separation from the free antigen directly measured (H. Keilacker, I. Ryazanovsky, M. Ziegler, D. Michaelis, K.- P. Woltanski, W. Besch: insulin antibodies in diabetic stability, insulin requirement and duration of insulin treatment, Horm. Meta. Res. 14 [1984], 227) or the antigen is immobilized on a solid phase (eg sepharose or plastic). After incubation with the sample, mostly serum, the antibodies specifically bound to the solid phase via the antigen are then detected as an antigen by means of a labeled second antibody (M. Koch, C. Frangois-Goard, F. Sodoyez-Goffaux, J. -C. Sodoyez: Semi-quantitative assessment of anti-insulin total IgG and IgG subclasses in insulin-immunized patients using a highly sensitive immunochemical micromethod., Diabetologia 29 [1986], 720). In this case, the prevention of non-specific immunoglobulin binding is often very problematic, whereby the sensitivity and reliability of the antibody detection is reduced. This also applies to the detection of antibodies to cell or tissue-bound antigens such as to microorganisms, to isolated cells (M.Ziegler, B. Ziegler, P. Heinke, I. Ryazanovsky: Titre determination of autoantibodies [IGSA] against beta cell surface Fresenius Z. Analyt, Chem.330 [1988], 354) or to tissue sections (H. Gleichmann, GF Bottzazzo: Progress toward standardization of cytoplasmic iclet cell antibody assay, Diabetes 36 [1987], 578).

According to previous Nac.iweisverfahren the antigen specificity of autoantibody detection is possible only with the help of the pure antigen, provided that the antigen can be labeled or immobilized to a solid phase without losing its immunoreactivity.

If only one t rimplex antigen pool is available as a target, eg. As an organ extract or cell suspensions with different cell types, all binding antibodies are detected, regardless of their diagnostic relevance. In addition, the non-specifically bound immunoglobulins are also determined.

Object of the invention

The aim of the b'i invention is a reliable method for the detection and quantitative determination of specific, diagnostically relevant autoantibodies with low technical costs.

Explanation of the essence of the invention It is an object of the invention to provide an immunological detection method for specific autoantibodies in serum

and other bodily fluids to concomitantly bypass the non-specific binding of the immunoglobulins of the test proteins, the structure and class of antigens with which the monoclonal antibodies react may still be unknown.

The indicated disadvantages of the current state of the art for the specific determination of autoantibodies

be solved according to the invention in an advantageous manner by using monoclonal antibodies, the z. For example, on the diagnostic relevance for antigen specificity due to the competitive inhibition of their antigen binding by polygonal antibodies from patients; Heen were selected with certain diseases.

This method consists of using monoclonal antibodies with the specificity relevant for autoantibody determination

be immobilized by the bound to a solid phase complex antigen mixture only epitope specific and by

Preincubation, preferably in the period of 1 hour or simultaneous incubation to preferably 3 hours,

for example, with human sera or other sample material, reduced in their binding, or completely inhibited, depending on the prevalence and concentration of polyclonal antibodies of identical epitope specificity in the test sample.

The binding or specific displacement of the monoclonal antibody is directly measurable when it is in labeled form

is used. Otherwise, the antigen-bound specific monoclonal antibody will become monoclonal by one

Antibody-binding, labeled second antibody determines the bound to irrelevant antigens human Antibodies and the non-specifically bound serum immunoblotting are not detected. This will make a higher Specificity and at the same time higher sensitivity of autoantibody detection achieved. As target antigen preparations z. As organ or cell extracts, microorganisms, cell suspensions and Tissue thin sections are used. The specificity of the autoantibodies detected depends on the specificity of the human antibody. The procedure is

also suitable for detecting epitope-specific autoantibodies for immobilized pure antigens.

Ausführungsbeisplel

The inventive method will be explained in more detail below. According to the invention, specific autoantibodies are to be detected, for example, against beta cells in insulin-dependent (type 1) diabetes mellitus and against thyroid antigens in Graves' patients

 I.Beispiel

50000 cells are pipetted into each well of a 96-microtitre plate as a target antigen preparation of an insulin-producing rat insulinoma cell line and dried. In 20 wells each 50μΙ control sera of healthy donors and in the remaining 50 μΙ serum of freshly-manifested type 1 diabetics are pipetted in each case as three-dimensional determination. After preincubation at room temperature for 1 h, 50 μl of a murine monoclonal betacell antibody are added by pipette into each reaction vessel. After thorough mixing, the mixture is incubated again at room temperature for 3 h. The complete plate is emptied and washed twice. To detect the bound monoclonal betazeil antibody, ΙΟΟμΙ of a peroxidase-labeled antibody against mouse immunoglobulin is added. After 90 minutes incubation, the plate is emptied, washer, and for color development for 30 min at room temperature '- ~ ^! t 100 μΙ o-phenyldiamine / H2O ₂ incubated and stopped with 100μ11 mol / l H ₂ SO ₄ . The color absorbance is at 492 nm in SUMAt. System measured. Serum samples that cause a reduction in absorbance to less than x-3D of the control sera are considered to be autoantibody-positive.

2nd example

An islet cell suspension containing 5 × 10 ⁴ cells in 50 μl cell culture medium is incubated for 1 h with 50 pm serum from control persons or freshly manifested type 1 diabetics in a test tube. After addition of 50 μM of a monoclonal beta-silica surface antibody and further incubation for 2 h at room temperature, the cells are washed twice and incubated with fluorescein isothiocyanate-labeled autoantibodies against mouse immunoglobulin and washed. The cells are transferred to slides and the percentage of surface fluorescence positive cells are counted on the fluorescence microscope.

Serum samples that cause a reduction to less than x-3SD fluorescent cell portion of the control sera are considered positive autoantibody.

3rd example

Glass tubes are coated on the bottom with rat insulinoma cells and 50 μM of a murine monoclonal beta cell antibody in cell culture medium and 50 μM serum of control or freshly-manifested Type 1 diabetics are incubated overnight at 4 ° C. After decanting the supernatant and washing the cells, they again 30min with 100μΙ ^m J-anti-mouse immunoglobulin are incubated at room temperature.

After washing, the bound radioactivity is measured in the gamma counter. Serum samples that cause a reduction in radioactivity to less than x-3 SD of control serum samples are considered positive for autoantibodies.

4th example

Microtiter plates are coated with thyroid membranes as a target antigen preparation. In 20 wells each 50μΙ control sera of healthy blood donors and in the remaining 50 μΙ serum of patients with immunogenic thyroid disease are pipetted in triplicate. After 1 h preincubation at room temperature 50μΙ

of a murine monoclonal thyroid antibody is pipetted into each reaction vessel. After thorough mixing, the mixture is incubated again at room temperature for 3 h. The complete plate is emptied and washed. For quantitative detection of the binding or displacement of the monoclonal antibodies, if they are not labeled themselves, they are visualized by the indirect method with a POD-labeled antibody against mouse immunoglobulin with the usual color development (see Example 1).

Serum samples that cause a reduction in absorbance to less than χ - 3SD of control sera are considered positive for autoantibodies.

Claims (7)

1. Immunological Nan! iweisverfahren for specific autoantibodies in the blood serum or other body fluids, characterized in that these autoantibodies by preincubation with complex antigen preparation and subsequent or simultaneous incubation with a monoclonal indicator antibody inhibit this monoclonal antibody in its specific binding to the antigen preparation. 2. Immunological detection method for specific autoantibodies according to claim 1, characterized gekonnzeichnet that with the same complex antigen preparation from a polyclonal antibody pool in separate reaction vessels with different monoclonal antibody populations with different epitope specificity of the detection. 3. Immunological detection method for specific autoantibodies according to claim 1, characterized in that are used as complex antigen preparations unpurified organ extracts or cell suspensions, microorganisms or Gewebedünnschnitte with different cell types. 4. Immunological detection method for specific autoantibodies according to claim 1, characterized in that the pre-incubation is preferably carried out for 1 hour and the incubation with the monoclonal antibody, preferably for 3 hours. The immunological detection method for specific autoantibodies according to claim 1, characterized in that the relevant antibody population in the test sample is detected directly by displacement of the formation of the labeled monoclonal antibody, and there is no species-dependent limitation in the application of this method. Immunological detection method for specific autoantibodies according to claim 5, characterized in that the inhibition of binding of the specific monoclonal antibodies by a labeled second antibody directed against the immunoglobulins of the species of origin of the monoclonal antibodies is detectable, the test sample and the monoclonal antibody Antibody may not be the same species. 7. An immunological detection method for specific autoantibodies according to claim 1, characterized in that the autoantibodies are determinable against any antigenic structure, independently of the substance class.