Homogeneous antibodies production

Monoclonal Antibodies (mAB). Exposure to antigens such as those found on bacteria or viruses triggers the production of a lai e number of different antibodies, each of which is specific for a particular molecular determinant on an organism. In the 1960 s, it was discovered that persons with multiple myeloma, a cancer of plasma cells, produced large quantities of homogeneous antibodies. 

A portion of this chapter will be concerned with antibodies to bacterial vaccines, particularly killed streptococci and pneumococci. Such antigens are of unique value for the production of homogeneous antibodies because of the very high concentrations of precipitable antibody which can be induced in experimental animals, as well as the frequent appearance of uniform subpopulations. Serum concentrations exceeding 15 mg/ml are common and values as high as 60 mg/ml have been reported (e.g., 4). Not all such sera possess a readily demonstrable homogeneous component when the latter is present it may account for a small or a large fraction of the antibody. 

One approach to the production of homogeneous antibody involves adoptive transfers of limited numbers of cells into irradiated syngeneic mice. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

The production of MAbs offers investigators a homogenous collection of antibodies that could bind selectively to a specific antigenic determinant with the same affinity. In addition to protein isolation and diagnostic techniques, MAbs have contributed greatly to RIAs. 

Factor IX may also be purified by immuno-alfinity chromatography, using immobilized anti-IX murine monoclonals. Purification to homogeneity is particularly important in the case of recombinant products. At least one monoclonal antibody has been raised that specifically binds only to factor IX, which contains pre-bound Ca + (i.e. the Ca +-dependent conformation of factor IX). Immobilization of this antibody allowed the development of an immuno-alfinity system in which factor IX binds to the column in the presence of a Ca -containing bufier. Subsequent elution is promoted simply by inclusion of a chelating agent (e.g. EDTA) in the elution buffer. 

A direct comparison of catalysis of olefin epoxidation with a homogeneous chemical catalyst (Mn salen), an enzyme (CPO), and an antibody resulted in sufficiently high enantioselectivity for all three catalysts, a higher turnover number for the enzyme, and a slightly higher substrate/catalyst ratio for the homogenous catalyst. Criteria for comparison should be quantitative and include catalyst lifetime as well as volumetric productivities, but have been found to depend on the different needs of laboratory synthetic chemists, who need a broadly specific catalyst quickly, versus those of process chemists, who often control catalyst availability and can allow narrow specificity (provided their substrate is accepted) but need high productivity. 

In summary, only a few therapeutic polyclonal antibodies are still marketed today. The main advantages of mAbs are their high specificity towards the target and the capability of an unlimited production of these homogeneous biological molecules. In future, new antibody or antibody-derived pharmaceuticals will be developed and can be expected to have favorable efficacy, a lack of immunogeni-city, and appropriate pharmacokinetics such that they can be used to treat intracellular medical disorders. 

In conclusion, studies on isolated cells allow measurements on homogeneous cell populations under well-defined conditions and can aid in the interpretation of metabolic changes seen by NMR in the same or similar cells in the intact animal. In some cases the cells may be a valid system for study in their own right. For example, there have been several NMR studies of commercially important mammalian cell lines which are used in the biotechnology industry for the production of various monoclonal antibodies and recombinant proteins with therapeutic or analytical applications (Mancuso et al., 1990 Gillies et al., 1991). 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is... 

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