Production of appropriate antibodies

On the day of immimization, add methylated BSA so that its concentration by weigjit is equal to that of the DNA, i.e. add 15 jU of methylated KA solution (1% (w/v) in water) per ml of DNA solution. This usualty results in a fine precipitate of DNA-protein complex.  

Mix the resulting suspension with an equal volume of Freund s adjuvant-complete for first, incomplete for subsequent immunizations. 

Volumes and routes of administration of immunogen will probably depend upon immunization protocols already in use. Protocol 1 is satisfactory for rats. 

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Depending on the use of the mAbs, certain adaptations may be required for their preparation. When large quantities of mAbs are required, in vivo production of the antibody in ascitic fluid is not practical, because it will require the use of a large number of animals. Thus, it is often easier to cultivate the antibodies in an appropriate in vitro culture medium. However, given the strict nutritional requirements of the hybridomas and their fragility in the face of osmolality, pH variations, and the accumulation of metabolites, the production of large quantities of antibodies in vitro will necessitate special care. 

The diversity of the immune response is impressive in that any foreign substances (antigens), small molecules or macromolecules, can elicit productions of appropriate proteins (antibodies) in response to diverse structures of antigens. In addition, the antibody response shows remarkable specificity. The specificity of the antibody-antigen interaction might be regarded as a model for molecular recognition. 

Antibody-based probes (immunosensors) offer another highly specific probe technology, since antibodies recognize very specific sites or cellular components (epitopes). Antibodies specific for any microbe can be made if the microbe can be obtained in pure culture. These must be screened for binding characteristics, that is, binding affinity, on- and off-rates, and epitope recognized. The production of monoclonal antibodies requires significantly more time and effort in the development of hybridoma cell lines with appropriate characteristics. It is, therefore, desirable to provide... 

In 1975, the first successful production of MAbs was reported (44). By fusing normal antibody-producing cells with a B-ceU tumor (myeloma), hybridoma cell lines resulted which produced antibodies having a specificity to only one deterrninant on an antigen ie, all the antibodies produced from the cell line are identical. These studies resulted in a standard approach to MAb production. In this approach, the hybridoma cells are produced in large quantities in culture and screened to select specific clones producing the desired MAb using an appropriate assay. The selected clones are then expanded in culture (or in animals), the cells are collected, and the MAbs are extracted and purified. 

Secondary antibody and determination. A secondary antibody labeled with an enzyme is added which binds to the primary antibody that is bound to the coating antigen. If the primary antibody were produced in a rabbit, an appropriate secondary antibody would be goat anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (or another enzyme label). Excess secondary antibody is washed away. An appropriate substrate solution is added that will produce a colored or fluorescent product after enzymatic conversion. The amount of enzyme product formed is directly proportional to the amount of first antibody bound to the coating antigen on the plate and is inversely proportional to the amount of analyte in the standards. 

The presence of foreign protein in the medium of plant cultures does not necessarily mean that all or even most of the product can be recovered from the medium. In many expression systems where an appropriate signal sequence has been used, considerable amounts of foreign protein remain within the plant cells and/or tissues. For example, in a comparison of IgG antibody production in tobacco cell suspension and hairy root cultures, a maximum of 72% of the total antibody was found in the medium of the suspension cultures whereas only 26% was found in the medium of the hairy root cultures [17]. This result could indicate that secretion and/or transport across the cell wall was slower in the hairy roots alternatively, it could indicate poorer stability of the secreted protein in the hairy root medium. If foreign proteins are to be purified from the medium, improved secretion and extracellular product stability are desirable. 

If the product is an antibody, then it is essential to distinguish the immunoglobulin product, e.g., mouse IgG, from any media immunoglobulin components, e.g., bovine IgG. Lucas et al.16 developed an immunoassay to measure nanogram quantities of bovine IgG in the presence of a large excess of a structurally homologous protein, mouse MAb. The bovine IgG was a contaminant that copurified with the product from a protein A column. For the bovine IgG assay, whole IgG and protein A-purified IgG reacted differently in the assay. It is important to evaluate these types of assays for cross-reactivity. For other media components, such as chemicals or antibiotics, ELISA is probably not the most appropriate method due to the low immunogenicity of chemicals. Techniques such as HPLC would be better to detect these chemical components. 

Many biopharmaceutical preparations are heterogeneous and may be difficult to fully characterise. Certain fractions of a preparation may have different biological activity or kinetics than the intended product. It is important that such fractions are appropriately qualified. The proportions of these fractions may be altered when production changes are made or they may be different between similar products produced by different manufacturers. Because of their proteinaceous nature and their novel mechanisms of action, all preclinical and clinical development steps must be re-evaluated. For pharmacokinetic studies, blood concentrations should be measured by specific analytical techniques (most often ELISA), which quantify the active protein and not one of its fragments or inactive forms, such as antigen-antibody complexes. For PK-PD studies of monoclonal antibodies, relevant biomarkers are most often circu-... 

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