Chemistry: analytical and immunological testing – Biospecific ligand binding assay
Patent
1986-05-29
1989-06-27
Schain, Howard E.
Chemistry: analytical and immunological testing
Biospecific ligand binding assay
436506, 436518, 436530, 436548, 436526, 436806, 436808, 436813, 436804, 436544, 436545, 436546, 435 7, 435810, 530387, 530395, 424 858, G01N 3353, G01N 33533, G01N 33534
Patent
active
048430196
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to acino-fetal differentiation proteins associated with cancer of the pancreas, to their purification, to a specific antiserum and the preparation thereof, to monoclonal antibodies specific for these proteins and the process for preparation thereof, and also to the compositions for diagnostic use and therapeutic use containing them.
The demonstration of the presence, in extracts of fetal pancreas, of antigens similar to those of pancreatic tumor cells is known.
Human oncofetal pancreatic antigens have been identified, having a molecular weight of 40 KD (POA-1) [banwo o., Versey J. and Hobbs J. R., New Oncofetal antigen for human pancreas. Lancet, 1: 643-645, 1974 ; Hobbs J. R., Knapp, M. L. and Branfoot A. G. Pancreatic oncofetal antigen (POA): its frequency and localisation in humans. Oncod. Biol. and Medicine, 1: 37-48, 1980], 800 and 900 KD (POA-2) [Gelder F. B., Resse C. J., Moossa A. R., Hall T. and Hunter R. Purification partial characterization and clinical evaluation of a pancreatic oncofetal antigen. Cancer Res. 38: 313-324, 1978] and 1,000 KD (PCAA) (Shimano T., Loor R. M. Papsidero L. D. et al. Isolation, characterization and clinical evaluation of a pancreas cancer associated antigen. Cancer., Supplement, 47: 1602-1620, 1981]. However, none of these antigens is specific to cancer of the pancreas, since they occur in tissues other than pancreatic cancerous tumors.
The present invention relates to acion-fetal (sic) differentiation proteins associated with cancer of the pancreas, these proteins being mannoside-containing glycoproteins, of average apparent molecular mass chosen from: 120 KD, 94 KD, 75 KD and 58 KD.
The present invention also relates to a method for preparing these proteins, wherein a soluble homogenate of human fetal pancreas is prepared and wherein a fraction consisting of mannoside-containing glycoproteins is isolated.
The method for preparing these antigens comprises the following stages: concanavalin A coupled to a gel in order to bind the said proteins,
The preparation of the homogenate is described in Example 1.
Young fetuses of gestational age less than 6 months, and preferably less than 5 months, are used because the proteins according to the invention do not appear in the adult pancreas and because rabbits injected with pancreas from fetuses older than 6 months did not produce detectable antibodies.
In a particular embodiment, a gel of Sepharose 4B coupled to concanavalin A, marketed by Pharmacia, was used. The unbound fraction is removed, and the bound fraction is eluted using a specific ligand, e.g. 0.2 M .alpha.-methylmannoside. The eluted bound fraction is then dialyzed by a conventional technique and the proteins are separated, for example by preparative electrophoresis. The peaks corresponding to the proteins according to the invention have been identified beforehand by qualitative electrophoresis.
The acrylamide gel fractions charged with proteins according to the invention are then removed and the proteins are extracted therefrom by electroelution in a dialysis tube against PBS phosphate buffer (saline solution 0.15 M NaCl buffered to pH 7 with potassium phosphate buffer, 0.01 M final concentration). The proteins are dialyzed against distilled water and lyophilized. A characterization of the proteins according to the invention is described in Example 6.
To separate the differentiation proteins, the techniques of immune binding to the monoclonal antibodies can also be used.
The present invention also relates to an antiserum against the antigens according to the invention.
By application of the blotting technique (after acrylamide gel electrophoresis--PAGE--the proteins which have migrated into the gel are transferred electrophoretically to a nitrocellulose sheet; this sheet is then treated as in the NIF technique), this antiserum recognizes, in the extracts of fetal pancreas, 4 constituents of average apparent molecular mass 120, 94, 75 and 58 KD (to within 10%).
The present invention also relates to a method for preparing this antiseru
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Burtin Pierre
Escribano-Crespo Maria J.
Centre National de la Recherche Scientifique
Draper Garnette D.
Schain Howard E.
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