The General Hospital Corporation

Massachusetts General Hospital Corporation

Gerald P Budzik

Patricia K Donahoe

David A Swann

Meredith Mudgett-Hunter

In a method for preparing a monoclonal hybridoma which secretes antibodies against an immunogenic material, comprising sensitizing an appropriate source with an immunizing amount of a preparation of the immunogenic material, obtaining from the source lymphocytes having immune activity against the immunogenic material, and fusing the lymphocytes with an appropriate cell line to thereby form mixed hybridomas, the improvement wherein: the immunogenic material is present in the immunizing preparation in an amount not larger than 10% by weight, and wherein, after formation of the mixed hydridomas but prior to cloning, the method comprises the steps of: raising ascites fluid with the mixed hydridomas, then detecting immune specificity against the immunogenic material in the ascites raised fluid; and thereafter cloning the ascites raised cells which show immune specificity against the material.

Monoclonal antibody to mullerian inhibiting substance

A process for purifying Mullerian Inhibiting Substance (MIS) from testicular tissue which comprises incubating testicular tissue in a culture media in the presence of a protease inhibitor in order to extract MIS from the tissue, chromatographing the extract on an anionic exchange resin followed by chromatographing the biologically active product obtained therefrom on a cationic exchange resin; the biologically active material from the cationic exchange resin is chromatographed by lectin affinity chromatography on wheat germ lectin and then either chromatographed by lectin affinity chromatography on concanavalin A or by triazinyl dye affinity chromatography on a appropriate triazinyl dye; MIS is obtained in 8,000-fold and 15,000-fold purification over original testicular tissue.

Purified Mullerian Inhibiting Substance and method of use

In a method for preparing a monoclonal hybridoma which secretes antibodies against an immunogenic material, comprising sensitizing an appropriate source with an immunizing amount of a preparation of the immunogenic material, obtaining from the source lymphocytes having immune activity against the immunogenic material, and fusing the lymphocytes with an appropriate cell line to thereby form mixed hybridomas, the improvement wherein: the immunogenic material is present in the immunizing preparation in an amount not larger than 5% by weight, and wherein, after formation of the mixed hybridomas but prior to cloning, the method comprises the steps of: raising ascites fluid with the mixed hybridomas, then detecting immune specificity against the immunogenic material in the ascites raised fluid; and thereafter cloning the ascites raised cells which show immune specificity against the material.

Method of preparing hybridomas and of purifying immunogenic materials

A process for purifying Mullerian Inhibiting Substance (MIS) from testicular tissue which comprises incubating testicular tissue in a culture media in the presence of a protease inhibitor in order to extract MIS from the tissue, chromatographing the extract on an anionic exchange resin followed by chromatographing the biologically active product obtained therefrom on a cationic exchange resin; the biologically active material from the cationic exchange resin is chromatographed by lectin affinity chromatography on wheat germ lectin and then either chromatographed by lectin affinity chromatography on concanavalin A or by triazinyl dye affinity chromatography on an appropriate triazinyl dye; MIS is obtained in 8,000-fold and 15,000-fold purification over original testicular tissue.

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Waltham, MA, United States of America

Gerald P Budzik