Monoclonal antibodies specific for membrane-associated antigens.

0001The invention relates to monoclonal antibodies that bind to defined membrane-associated antigens. These antibodies can be used as diagnostic agents or as drug carriers.

0002The technique of immunization with defined, isolated antigens and the production of antibodies against such antigens is known. It is also known to immunize with unpurified antigen material and to select those antibodies which recognize a specific component of such an antigen mixture.

0003When attempting to induce such antibodies, it was possible to select monoclonal antibodies which react under non-reducing conditions with the following protein antigens: antigen 1: MW 33 KD ± 3, antigen 2: MW 134 KD ± 3, antigen 3: MW 80 KD ± 3, antigen 4: 55 KD ± 3, antigen 5: 60 KD ± 3, antigen 6: 54 KD ± 3, antigen 7: 260 KD ± 3, antigen 8: not determinable, antigen 9: not determinable, antigen 10: MW 143 KD ± 3, 119 KD ± 3, antigen 11: MW 178 KD ± 3, antigen 12: MW not determinable, antigen 13: MW 34 KD ± 3, antigen 14: MW 195 KD ± 3, antigen 15: MM 44 KD ± 7, antigen 16: MW 43 KD ± 3, antigen 17: MW 130 KD ± 3. Die on the antigens 1-17 recognized by the AK 1-17 epitopes are differently sensitive to treatment with dithiothreitol (50 mmol / l, 2 hours, 37 ° C): The epitopes of Agl, 2 and 15 are changed by the above treatment that of Ag4, 10 and 14 remain unchanged.

0004The in vitro specificities of AK 1-17 are shown in Table I.

0005The invention relates to monoclonal antibodies which recognize an antigen (Agl) with a molecular weight of approximately 33 KD ± 3 or one<ul id="ul0001" list-style="none"><li>Antigen (Ag2) with a molecular weight of approximately 134 KD ± 3 or one</li><li>Antigen (Ag3) with a molecular weight of approximately 80 KD ± 3 or one</li><li>Antigen (Ag4) with a molecular weight of approximately 55 KD ± 3 or one</li><li>Antigen (Ag5) with a molecular weight of approximately 60 KD ± 3 or one</li><li>Antigen (Ag6) with a molecular weight of approximately 54 KD ± 3 or one</li><li>Antigen (Ag7) with a molecular weight of approximately 2G0 KD ± 3 or one</li><li>Antigen (Ag8) with an undetermined molecular weight or a</li><li>Antigen (Ag9) with an undetermined molecular weight or a</li><li>Antigen (Ag10) with a molecular weight of approximately 143 KD ± 3, and 119 KD ± 3 or one</li><li>Antigen (Agll) with a molecular weight of approximately 178 KD ± 3 or one</li><li>Antigen (Ag12) with an undetermined molecular weight or a</li><li>Antigen (Ag13) with a molecular weight of approximately 34 KD ± 3 or one</li><li>Antigen (Ag14) with a molecular weight of approximately 195 KD ± 3 or one</li><li>Antigen (Ag15) with a molecular weight of approximately 44 KD ± 7 or an antigen (AglG) with a molecular weight of approximately 43</li><li>KD ± 3 or an antigen (Agl7) with a molecular weight of approximately 130 KD ± 3.</li></ul>

0006Antigens 4,8,9,10 are mycoplasma antigens that are present on cell lines described in the table. Human in vitro cultivated cell lines, cell extracts or extracts from human tissues serve as the immunogen for inducing these antibodies. Permanent human cell lines are preferred, in particular the CaLu-1, Chago, Oat 75, PaTu II and Bewo cell lines. Agl - Ag17 can also be used to generate AKl - AK17.

0007Mammals, preferably mice, are 1 x 10^{6 -} 10⁸ Cells, but preferably 10⁷ Cells of such a cell line were immunized intraperitoneally (day 0-120, but preferably on days 0 and 7) and after 1-150 days, but preferably on day 11, the spleen cells from such animals with the X63 Ag 8653 cell line (The Journal cf Immunology 173, 4, 1548-1550, 1979) (Nature 256, 495-497, 1975).

0008The hybridomas that develop after 3 weeks are tested to determine whether they contain antibodies of the desired specificity. In this case, a range of 30 in vitro cultured cell lines, human peripheral blood cells and human bone marrow was determined using indirect immunofluorescence (Dehring Inst. Mitt. 59, 64-70, 1976) and cell sorter analysis (Acta Cytol. 19, 374 -377, 1975), (Proc. Natl. Acad. Sci. USA 77/8, 4914-4917, 1980) tested for reactivity with the antibodies.

0009Surprisingly, among the hybridoma residues tested were some that contained antibodies of the interesting specificities described above. The hybridomas secreting these antibodies were cloned using a micromanipulator and the monoclonal antibodies obtained from these hybridoma clones were used to immunochemically characterize the antigen they recognized. For this purpose, cell ghosts (Hybridoma, 1, 4, 413-421 (1982)), which are obtained from tissue culture cells, are solubilized by means of detergent, ultracentrifuged and the antigen mixture obtained in this way under non-reducing conditions in SDS-polyacrylamide gel (10 g / 100 ml acrylamide / 0.026 g / 100 ml bisacrylamide separated electrophoretically (Nature, 127, 680-685 (1970)).

0010Then all the antigens separated in this way are transferred from SDS gel to nitrocellulose filter by means of electroblotting. (Proc. Natl. Acad. Sci. USA, 76, 4350-4354 (1979)). The antigens immobilized on the nitrocellulose filter are then reacted with the specific antibody (Hoppe-Seylers Z. Physiol. Chem. 363, 1133-1140, (1982)). The binding of this antibody is detected by reaction with¹²⁵J protein _A and autoradiography of the filter. The molecular weight is determined in comparison to commercially available markers.

0011Preparative purification of the corresponding antigens is carried out by affinity chromatography.

0012Nitrocellulose paper (15 x 15 cm) (Hoppe-Seylers Z.

0013Physiol. Chem. 363, 1133-1140, (1982)) is incubated after moistening with phosphate-buffered saline (PBS pH 7.2) with purified monoclonal antibody protein (1 hour, 4 ° C, 30 ml, 1-100 µg protein / ml).

0014The unbound protein is then removed by washing in 500 ml of PBS. After incubating the filter for 1 hour at 40 ° C. in 3 g / 100 ml BSA (ox serum albumin) 0.05 g / 100 ml Tween 20 in PBS pH 7.2 (blocking), the filter is washed with PBS three times with unpurified Cell extracts, which are dissolved in 0.5% sodium deoxycholate, 20 mM phenylmethylsulfonyl fluoride in PBS, incubated for 1 h at 4 ° C.

0015The unbound material is then removed by washing 3 times with PBS. The specifically bound antigen is obtained by incubating the filter in 1-9 M NH₄SCN prefers 6 M NH₄SCN detached in PBS for 5 to 30 minutes, preferably 15 minutes at 4 ° C.

0016The antigen purified in this way can be removed after removal of the NH₄SCN (gel chromatography, dialysis) can be used as an immunogen or for other purposes.

0017The monoclonal antibodies characterized by their reactivity with Agl and Ag2 can be used to differentiate between cells derived from solid human tissue and from animal cells as an in vitro diagnostic. Furthermore, they could be used as a positive control for human tissue in immunohistology and for the differentiation of animal tissue.

0018AK1 to AK 17 can also be used to bring specific active ingredients to the site of action, e.g. B. in autologous bone marrow transplantation. The use of these antibodies for the detection of the corresponding Ag in body fluids represents a diagnostic application of these antibodies. The antibodies can be used in concentrations of 0.01 mg to 1 mg / ml.

0019<tables id="tabl0001" num="0001"><img file="EP0141079A2_D0001.tif" /></tables><tables id="tabl0002" num="0002"><img file="EP0141079A2_D0002.tif" /></tables>

0020<ul id="ul0002" list-style="none"><li>+ = significant positive reaction in the indirect immunofluorescence test, in the radioimmunoassay and in the cytofluorometric analysis.</li><li>± = weak positive reaction with part of the tested population</li><li>- = no significant reaction</li></ul>