Antibodies product modifications

Some synthetic carriers actually are designed to have low immunogenicity on their own to minimize the potential for antibody production against them. When a hapten is coupled to these molecules, the immune response is directed principally toward the modification, not at the carrier. This design approach guides most of the immune response toward the desired target and minimizes the production of carrier-specific antibodies. 

Burnet, F.M. (1957). A modification of Jeme s theory of antibody production using the concept of clonal selection. Aust. J. Sci. 20,67-69. 

The initial steps of solubilization, DEAE chromatography and gel filtration were slight modifications of reported procedures [19]. The pooled fractions from a Sephacryl S-200 (Pharmacia) column were applied to a Mono Q HR5/5 FPLC column (Pharmacia) and eluted in a 20 ml gradient from 0-350 mM NaCl in 0.25 M sucrose-20 mM Tris-HCl pH 7.3. Fractions of 1 ml were collected and assayed for binding of NAA-1-[ C] (61 mCi/mmol, Amersham) by one of three methods [20]. The most active fractions were pooled, desalted and lyophilized. This preparation (approx. 50% receptor) was used either for monoclonal antibody production or was fully purified by native PAGE in a neutral pH discontinuous system [3]. The gel was briefly electroblotted (5 min, 10 mA) to nitrocellulose and the small fraction of transferred proteins visualized by rapid staining [8]. This blot was then used to locate precisely the bulk protein bands remaining in the gel. 

Antibodies were first expressed in transgenic plants in 1989. Since then various antibody fragments and domains have been produced in plant hosts as well as full-length and multimeric antibodies. The most popular host species for this work has been the tobacco plant, Nicotiana, although com and soybeans have also been utilized. There is no apparent reason why other plants could not be used. The value of using plants for monoclonal antibody production include the absence of animal pathogens, the ease of genetic manipulation, the ability of post-translational modification, and the potential for scale-up to an economic production process. 

In 1967, Heidelberger, Stacey et al. reported the purification, some structural features, and the chemical modification of the capsular polysaccharide from Pneumococcus Type I. Difficulties of direct hydrolysis of the polysaccharide were overcome and it was possible to identify some of the fragments in the hy-drolyzate. At least six products resulted from nitrous acid deamination. Two were disaccharides, which were identified, and sequences of linked sugar units were proposed. As modification of the polysaccharide decreased the amounts of antibody precipitated by anti-pneumococcal Type I sera, the importance of the unmodified structural features in contributing to the specificity of the polysaccharide was indicated. 

The goal of most protein modification or conjugation procedures is to create a stable product with good retention of the native state and activity. Ideally, any derivatization should result in a protein that performs exactly as it would in its unmodified form, but with the added functionality imparted by whatever is conjugated to it. Thus, an antibody molecule tagged with a fluorophore should retain its ability to bind to antigen and also have the added functionality of fluorescence. 

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT... 

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