Control y balance de liquidos administrados y eliminados
Technical Field The present invention relates to the branch of immunology and human medicine particularly with the generation and selection of monoclonal antibody mAb against oligosaccharide sequence sialic acid N-glycolylated-galactose-glucose, which can be used for diagnosis and treatment of certain neoplastic diseases.
The oligosaccharide structures can be found forming part of glycoproteins and glycolipids, they are either present in normal and pathological tissues. Frequent changes in the aberrant glycosylation are: Gangliosides are glycosphingolipids that the sialic acid containing in their structure and are characterized by being present in most vertebrate cells.
These molecules are found in normal tissues and may have a higher expression in the tumors, with a different organization and conformation on the surface of malignant cells Hakomori, SH,Cancer Res 45.
The humoral immune response carbohydrate antigens is generally of the IgM isotype. The oligosaccharide sequences attached to lipids are generally less immunogenic than the glycoproteins.
The use of glycolipids as immunogen requires therefore its coupling to carrier proteins or their incorporation to liposomes or bacterial strains such as Mycobacterium tuberculosis or R strain Salmonella minnesota.
The response generated against Gangliosides is thymus-independent and this aspect has been reported by Livingston et al repeatedly Livingston, PO et control y balance de liquidos administrados y eliminados,Proc Natl Acad Sci 84, ; Livingston, PO et al, Cancer res. Antibodies generated against specific gangliosides, when they have been studied in the serum of different species possess fundamental characteristics low affinity, considerable cross reactivity and short life Livingston, PO,Immunology and Allergy Clinics of North America, 11, ; Portoukalian, J.
The expression of the N-glycolylated control y balance de liquidos administrados y eliminados in the olygosaccharides is common in normal and pathological tissues of all the species of vertebrates, except for chickens and humans, which are only found in tumor and fetal tissues. The normal tissues of these two last species posses only the N-acetylated variant Nishimakit et al,J.
Immunology,; Higashi H. Studies oligosaccharide composition in some human tumors showed the presence of the N-glycolylated form both in glycolipids glycoproteins of tumor cells from melanoma Control y balance de liquidos administrados y eliminados, Y, et al.
Dermatol res 3 ; Biochem Control y balance de liquidos administrados y eliminados 2. Dermatol 11as well as in colon tumors, especially in glycolipids, Miyoshi, I. Additionally, studies were performed to verify the presence of the N-glycolylated ganglioside in some tumor samples liver, teratoma, lymphoma, etc, Kawai T.
The generation of monoclonal antibodies against the N- glycolylated gangliosides has provided until now, antibodies of IgM isotype that generally recognize more than one gangliosides molecule, for example human monoclonal antibodies M and M Control y balance de liquidos administrados y eliminados K.
Other authors have reported the generation of a specific species of antibodies against N-glycolylated gangliosides, always of IgM isotype, among which are the monoclonal antibody and HDone, against NGcGM 2 Shammai Y. However, Watarai Watarai, S. Chemist, Vol8, MAbs against gangliosides have been generated as HMA1 by.
Nagai and collaborators Nagai Y. Moreover in US 4, Yamasaki, M. Exp Med,. Passive immunotherapy with monoclonal antibodies against gangliosides has been used in clinical trials for the treatment of some tumors such as melanomas and neuroblastomas. Treatments in melanomas have been intralesional or systemic and although results seem promising, only a control y balance de liquidos administrados y eliminados number of patients had total or partial remissions Houghton, AN et al,Proc.
Oncol Cli Cancer, 24. Clin Oncol, 6. Saleh MN et al,cancer res. These antibodies showed effect in cytotoxicity studies, complement-mediated or cell-mediated Ravindramath MH et al,Inter.
Until now, all the monoclonal antibodies obtained against N-glycolylated gangliosides are of the IgM isotype and the toxicity they provoke is mediated by complement. IgM generally have low antigen affinity and it is difficult to use for purposes of diagnosis or therapy, when attempting to join them with radioactive isotopes. Although they are good complements fixers and guarantee a good cytotoxicity, the possibility of purification becomes quite cumbersome compared to IgG when this is done on a large scale.
Moreover monoclonal antibodies against glycoproteins N- glycolylated you have been little reported work and its purpose has been directed primarily for diagnostic purposes. It has also been published a monoclonal antibody designated JAM3, that recognizes the N-acetylated forms and N-glycolylated kD protein present on the surface of the cysts caused by the invasion of Entamoeba Avron, B.
The novelty of the present control y balance de liquidos administrados y eliminados to obtain a highly specific monoclonal antibody against the sialic acid oligosaccharide sequence gficolilado N- -Galactose-Glucose, present both in gangliosides and glycoproteins.
Furthermore, the fact that an immunoglobulin of IgGl isotype, gives the advantage to be more specific has more affinity for the molecules it recognizes, favoring Unexpectedly biological activity this antibody showed the capacity to provoke cellular death directly in tumor cells carrying control y balance de liquidos administrados y eliminados oligosaccharide sequence.
To obtain IgG MAbs against gangliosides the following immunization method is used. Mice or other mammalian species were immunized with vaccine preparations containing between 0.
Before and during the immunization period blood samples from the animals to obtain serum are taken with the purpose of monitoring the antibodies generated in the animals against the Ganglioside used as antigen, using any of the known immunoassay methods for detecting the antigen-antibody reaction Ag-Ab. Animals are immunized with various doses, between 2 and 8 at time intervals that can vary between 7 and 14 days. The administration is performed by subcutaneous or intramuscular route with volumes between 0.
Other possible immunization routes are intravenous use and intraperitoneal. The animals receiving this dose range show a specific response against the ganglioside with which they were immunized. For production of MAbs specific against the NGcGM3 control y balance de liquidos administrados y eliminados the mice with serum antibody titers against it received a new immunization with the vaccine preparation 3 days before obtaining the antibody-forming cells.
Spleen cells should preferably be chosen although others may also be used. These cells are fused with myeloma cells and provide it control y balance de liquidos administrados y eliminados the hybrid cells or hybridomas the ability to expand infinitely "in vivo" and "in vitro".
For this purpose, any of the known methods for cell fusion may be used. To determine the antibodies produced by the hybridomas an immunoenzymatic assay preferably used, although other immunoassay methods can also be used. The procedure of this assay is the recognition of hybridoma supernatants by gangliosides and the antigen-antibody binding can be visualized using a second antibody labeled by an enzyme, the antibody produced by the hybridoma under appropriate conditions and binds, in turn detected.
The hybridoma once selected is cloned at least 2 times for example, by limiting dilution. Mab obtained can be produced "in vitro" in an adequate culture medium, any of which are described in the prior art and further purified from the supernatant of said culture.
Another method of antibody production, involves injecting the hybridoma in animals for example, syngenic animals. The hybridoma causes formation of non-solid tumors that provide a large concentration of the desired antibody, blood level and in the peritoneal exudate of the host animal.
Purification of the monoclonal antibodies is from the ascitic fluid obtained by the inoculation of 0. The ascitic fluid is diluted by half in 1. Elution of MAbs is performed using citrate buffer 0, 14 M, pH 6. The visualization of the bands was performed by chemical development with Orcinol. Plates were plastican with poliisobutilmethacrilate solution and dried at room temperature overnight.
Then the monoclonal antibodies are incubated in the blocking solution. Upon completion washed with TFS and conjugated anti-immunoglobulin coupled to peroxidase mouse for 1 hour is added. It then proceeds to perform a new washing and then the solution with the enzyme substrate is added, to visualize the bands. Finally chemical development compared to the immune. Then cell viability studies are performed using the method of Trypan Blue. They can be counted dead cells by the effect of the antibody or by adding propidium iodide or other viability marker, this time in the flow cytometer by increased fluorescence in the red region.
Counts cell viability as described in the previous paragraph, were conducted to determine direct cytotoxicity or method of study of cell viability using the release of 51 Cr in the culture supernatant from the cells P3X63 Myeloma previously labeled with the radioactive isotope. MAbs generated can be used for both diagnosis and therapy, through labeling with a radioactive isotope, for example, 99mTc, Re and Re The method of labeling the antibodies by isotopes previously described by Schwarzn and Steinstrasser Schwarzn A.
To determine the potential usefulness of Mab, 10 mice were inoculated tumor was P3X63 and another 10 mice were used normal control, to which were not inoculated tumor. Monitoring of the control y balance de liquidos administrados y eliminados of the MAbs raised against the oligosaccharide sequence post-injection 24 hours at groups of 5 animals 5 healthy and 5 with tumorwhich are euthanized taking from the principal organs and is at 4 and the tumor, which are heavy and whose gamma emission quantified separately is the end of the experiment.
The monoclonal antibodies distributed in the healthy animals preferably in blood, liver and kidney while in the animals that had tumor was found in the above organs and preferably in the tumor at 24h. MAbs labeled with radioactive isotopes, as described in prior art, can be used to detect tumors where the oligosaccharide is expressed sequence. The radioactivity can be studied in whole body Gamma camera.
Images are acquired at 5 minutes, and 1, 3, 5, 24 and 48 hours after injection, the location of MAb appreciated only in the tumor and in the excretion organs. MAbs may also be coupled directly or indirectly to therapeutic agents such as drugs, radioisotopes, immunomodulators, lectins and toxins.
Within modifiers biological response immunomodulators that in some way can increase the destruction of the tumor by MAbs of the invention include lymphokines such as: Diagnostic purposes, immunohistochemical studies were conducted. Histopathology was evaluated in sections stained with hematoxylin and eosin.
The control y balance de liquidos administrados y eliminados were incubated with purified MAbs. Subsequently biotinylated serum was added anti-mouse and peroxidase streptavidin complex Dakopatts at room temperature. Between incubations sections were washed with Tris-buffered saline-HCl. The sheets were washed with running water contrasted with Mayers hematoxylin, covered with a mounting medium containing balsam and that coverslipped.
The reaction with the enzyme produces a brown-coloring carmelita. Fragments Fresh biopsies of pathological tissues were obtained within one hour following surgical excision, which were frozen and then cut, keeping frozen until study. The use of fetal tissues for the control y balance de liquidos administrados y eliminados is due to the association of gangliosides as oncofetal antigens has been repeatedly reported as well as the similarity of the expression of these molecules in fetal and tumoral tissue human Cahan, L.
The fetal tissue sections were obtained from fetus between 12 and 18 weeks. Of the tumors studied, lung and central nervous system with various etiologies were negative, as cuts normal human tissues, while melanoma and breast were all positive, and fetal tissues of the digestive system liverstomach, small and large intestine and the renal system. To demonstrate the antitumor effect of monoclonal antibodies against NGcGM3 ganglioside animals which are inoculated tumor having ganglioside white P3X63 myelomaare treated with the antibodies obtained.
Dosages may range from 0. MAbs generated can be administered parenterally by injection intravenous, intraperitoneal, intramuscular, subcutaneous, intracavity or transdermally. Chapman Hallwhere significant differences between the treated and control mAb, lower than 0. Each animal received 6 doses, the first control y balance de liquidos administrados y eliminados and 2 successive weekly every 14 days.
Antibody control y balance de liquidos administrados y eliminados were measured in the serum of animals using an indirect ELISA in Polysorp plates Nunc signature where gangliosides were immobilized following the methodology described below:. Then 50 ul of sera were diluted in the same buffer and incubated overnight at room temperature.
Wells were washed 4 times with Subsequently washing with PBS was repeated and 50 ul of a dilution of alkaline phosphatase streptavidin was added at a dilution line.