Rat monoclonal antibodies. I. Rapid purification from in vitro culture supernatants (original) (raw)
Related papers
Evaluation of protocols for purification of mouse monoclonal antibodies
Journal of Immunological Methods, 1986
Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-today reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.
Monoclonal Antibody Production
Monoclonal antibodies (mAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer. These antibodies are produced by cell lines or clones obtained from animals that have been immunized with the substance that is the subject of study. To produce the desired mAb, the cells must be grown in either of two ways: by injection into the abdominal cavity of a suitably prepared mouse or by tissue culturing cells in plastic flasks. Further processing of the mouse ascitic fluid and of the tissue culture supernatant might be required to obtain mAb with the required purity and concentration. The mouse method is generally familiar, well understood, and widely available in many laboratories; but the mice require careful watching to minimize the pain or distress that some cell lines induce by excessive accumulation of fluid (ascites) in the abdomen or by invasion of the viscera. The tissue-culture method would be widely adopted if it were as familiar and well understood as the mouse method and if it produced the required amount of antibody with every cell line; but culture methods have been expensive and time-consuming and often failed to produce the required amount of antibody without considerable skilled manipulation. However, culture methods are now becoming less expensive, more familiar, and more widely available
An ELISA has been set up for quantifying mouse monoclonal antibodies in culture supernatant. The assay includes rabbit anti-mouse IgG antibodies chromatographycally purified. This preparation was used as coating and as conjugated antibodies in the ELISA. The assay can detect IgGl with sensitivity of 0.2 ng/mL, IgG2a (0.85 ng/mL), IgG2b (0.13 ng/mL), and IgG3 (3.19 ng/mL) in culture supernatants. The effective working range was from subnanogram per mL quantities to 30 ng/mL by using a computer statistical program. Variation coefficient of ELISA was below 7%. Correlation estimates with a similar ELISA using commercial reagents were performed for each mouse antibody subclass. The assay was able to detect the four mouse mon-oclonal antibody subclasses in pure human serum as compared with the same ELISA using commercial an-tibodies. A 24-h pharmacokinetic profile of 1 patient treated with an IgG2a monoclonal antibody is presented.
The American Biology Teacher, 1984
As a graduate student 15 years ago, I worked in a lab with a student whose research involved purifying an antibody. At the time, pure antibody was difficult to come by. Immunizing an animal against a particular antigen could, of course, induce production of antibodies, but they were a heterogeneous collection with such similar properties that they were almost impossible to purify to homogeneity. Even if purification were achieved, the yield was so small as to be almost useless. Researchers tried to get around these problems by purifying antibodies from individuals with diseases in which large quantities of a single antibody were produced. Many used myelomas, which are tumors composed of B cells, the lymphocytes that make antibodies. Since the tumor was derived from a single cell, it secreted a single type of antibody. In our lab, antibody was purified from the psoriasis scales of a patient with a severe form of the disease. This antibody was more basic than most, which helped in purification, but it was still a difficult job (Lawrence, Tye, and Liss, Immunochemistry, January 1972). These strategies did succeed in providing researchers with relatively homogeneous antibody, and in making it possible to work out antibody structure. But since it was impossible to know the antigen that the antibody was directed against, the antibody's value as a research tool was limited. This situation changed in 1975 when George Kohler and Cesar Milstein developed the first antibodyproducing hybridoma. They began by immunizing a mouse against a specific antigen. The mouse's spleen cells then made antibody against that antigen. Since they were normal B cells, they had limited lifespans in culture, and so produced limited amounts of antibody. To overcome this problem, Kohler and Milstein
Semi-preparative purification and validation of monoclonal antibodies for immunotherapy in mice
Journal of Immunological Methods, 1994
A number of rat hybridomas were adapted to grow in RPMI containing either 5% IgG-depleted FCS or 1% serum-free Nutridoma. Alternatively, protein-free Ultradoma PF was used. Growth in these media allowed purification procedures to be used that are based on tangential ultrafiltration in combination with affinity chromatography on gels linked to protein G or anti-rat L chain coupled antibodies. The isolated antibody preparations were found to be pure and to consist of monomeric intact IgG. The yield and recovery of mAb using this procedure were found to be consistently high. These antibody preparations were analyzed for endotoxin contamination. Whereas during isolation endotoxin contamination increased, the endotoxin content per mg purified protein did not. Affinity chromatography on Detoxi-gel resulted in the efficient removal of this contamination and using this protocol the antibody preparations obtained were found to be of sufficient purity, activity and low endotoxin content to permit their in vivo use in animal models of immunotherapy
Journal of Chromatography A, 1985
A one-step chromatographic procedure was used to isolate rapidly mouse IgG monoclonal antibodies (mAbs) (IgG1, IgG2a, IgG2b) contained in ascites fluids and Fab fragments contained in papain-treated mAb suspensions. Chromatographic separations were performed on an anion-exchange Mono Q column connected to a fast protein liquid chromatographic (FPLC) system. Detection of mAb or their antigen binding fragments (Fab) in eluted peaks was performed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis together with a silver or a Coomassie Brillant Blue R 250 staining technique and solid phase radioimmunoassay with 12s I-labelled sheep anti-mouse antibodies directed against total immunoglobulins. Rapid assessment of the purity of isolated mAbs and their Fab fragments was performed by gel permeation chromatography on a TSK G 3000 SW column. Mouse mAbs and their Fab fragments were rapidly isolated (25 min), in a functionally active state, to a high degree of purity on the FPLC-Mono Q system compared to the time taken by other techniques.
Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell, in contrast to polyclonal antibodies which are made from several different immune cells. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope. Given almost any substance, it is possible to produce monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine. What are monoclonal antibodies? Antibodies are proteins produced by the B lymphocytes of the immune system in response to foreign proteins, called antigens. Antibodies function as markers, binding to the antigen so that the antigen molecules can be recognized and destroyed by phagocytes. The part of the antigen that the antibody binds to is called the epitope. The epitope is thus a short amino acid sequence that the antibody is able to recognize (Campbell NA, 1996). Two features of the antibody-epitope relationship are key to the use of monoclonal antibodies as a molecular tool. specificity-the antibody binds only to its particular epitope sufficiency-the epitope can bind to the antibody on its own, i.e. the presence of the whole antigen molecule is not necessary Structurally antibodies are proteins consisting of four polypeptide chains. These four chains form a quaternary structure somewhat resembling a Y shape. Figure 1 shows the three dimensional structure of immunoglobulin G, a typical antibody, and its schematic representation. Each B cell in an organism synthesizes only one kind of antibody. In an organism, there is an entire population of different types of B cells and their respective antibodies that were produced in response to the various antigens that the organism had been exposed to. However to be useful as a tool, molecular biologists need substantial amounts of a single antibody (and that antibody alone). Therefore we need a method to culture a population of B cells derived from a single ancestral B cell, so that this population of B cells would allow us to harvest a single kind of antibody. This population of cells would be correctly described as monoclonal, and the antibodies produced by this population of B cells are called monoclonal antibodies. In contrast, antibodies obtained from the blood of an immunized animal are called polyclonal antibodies.
Immunologic and pharmacologic concepts of monoclonal antibodies
Seminars in Nuclear Medicine, 1989
While monoclonal antibodies have solved many of the difficulties of using immunologic reagents for radioimmunodiagnosis and therapy, in the 13 years since their introduction a number of persistent problems remain, most notably a low yield of antibodyproducing cells from the fusion process, difficulty in obtaining high-affinity antibodies, and the potential immunogenicity of murine immunoglobulins (Igs). Several solutions are under development, including fusion techniques that enrich for cells producing desired antibodies, production of human-mouse chi-meric antibodies by recombinant DNA technology, and the generation of human monoclonal antibodies by promising new approaches. Until these upcoming methodologies are established, and to better direct their development and application, a sound understanding of the pharmacology of presently available native and modified monoclonal antibodies is crucial. Although much has been already determined in this area, a great deal of further clarification remains necessary.