Antibodies preparation

Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

Human immunoglobulin preparations from pools of a great number of people (>1,000) with assumed antibodies against common viruses are used as a means of passive immunization in acute infections. More specific antibody preparations with high titers from patients who recovered recently from a viral disease or were immunized against toxins are also available in... 

Remarkably, Brassica napus pollen was reported to have a 22 kDa cutinase that cross-reacted with antibodies prepared against F. solani f. pisi cutinase [134]. Although a 22 kDa and a 42 kDa protein that catalyzed hydrolysis of p-nitrophenyl butyrate were found in this pollen, only the former catalyzed cutin hydrolysis. Immunofluorescence microscopic examination suggested that the 22 kDa protein was located in the intine. Since the nature of the catalytic mechanism of this enzyme has not been elucidated, it is not clear whether this represents a serine hydrolase indicating that plants may have serine and thiol cutinases. The role of the pollen enzyme in controlling compatibility remains to be established. 

Such antibodies prepared initially against methylated bases (40), are becoming increasingly available for more complex modifications including PAHs (41-43). 

Based on the above principles, it might be assumed that a therapeutic protein obtained by direct extraction from human sources (e.g. some antibody preparations) or produced via recombinant expression of a human gene/cDNA sequence (e.g. recombinant human hormones or cytokines) would be non-immunogenic in humans whereas foreign therapeutic proteins (e.g. non-engineered monoclonal antibodies) would stimulate a human immune response. This general principle holds in many cases, but not all. So why do therapeutic proteins of human amino acid sequences have the potential to trigger an immune response Potential reasons can include ... 

Tumor necrosis factor Monoclonal antibody preparations y-Globulin preparations Hepatitis B surface antigen... 

Immunoassays employ monoclonal or polyclonal antibody preparations (Chapter 13) to detect and quantify the product (Box 7.1). The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (RIA) or enzymes (EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared with... 

Polyclonal antibody preparations have been used to induce passive immunity against a range of foreign (harmful) agents, and vaccines are used efficiently, and safely, to promote active immunization. Adjuvants are usually co-administered with the vaccine preparation, in order to enhance the immune response against the vaccine. 

Polyclonal antibody preparations have been used for several decades to induce passive immunization against infectious diseases and other harmful agents, particularly toxins. The antibody preparations are usually administered by direct i.v. injection. While this affords immediate immunological protection, its effect is transitory, usually persisting for only 2-3 weeks (i.e. until the antibodies are excreted). Passive immunization can be used prophylactically (i.e. to prevent a future medical episode) or therapeutically (i.e. to treat a medical condition that is already established). An example of the former would be prior administration of a specific anti-snake toxin antibody preparation to an individual before they travel to a world region in which these snakes are commonly found. An example of the latter would be administration of the anti-venom antibody immediately after the individual has experienced a snake bite. 

Antibody preparations used to induce passive immunity may be obtained from either animal or human sources. Preparations of animal origin are generally termed antisera , and those sourced from humans are called immunoglobulin preparations . In both cases, the predominant antibody type present is IgG. 

Although specific antisera have proven invaluable in the treatment of a variety of medical conditions (Table 13.1), they can also induce unwanted side effects. Particularly noteworthy is their ability to induce hypersensitivity reactions some such sensitivity reactions (e.g. serum sickness ) are often not acute, whereas others (e.g. anaphylaxis) can be life threatening. Because of such risks, antibody preparations derived from human donors (i.e. immunoglobulins) are usually preferred as passive immunizing agents. 

Although hypersensitivity reactions can occur upon administration of immunoglobulin preparations, the incidence of such events is far less frequent than is the case upon administration of antibody preparations of animal origin. As with all blood-derived products, the serum from which the immunoglobulins are due to be purified is first assayed for the presence of infectious agents before its use. 

The major polyclonal antibody preparations used therapeutically are listed in Table 13.1. These may generally be categorized into one of several groups upon the basis of their target specificities. These groups include antibodies raised against ... 

Table 13.1 Polyclonal antibody preparations of human or animal origin used to induce passive immunity against specific biological agents...

The antibody preparations could be administered unaltered or (more commonly) after their conjugation to radioisotopes or toxins. Binding of unaltered monoclonal antibodies to a tumour surface alone should facilitate increased destruction of tumour cells (Figure 13.4). This approach, however, has yielded disappointing results, as the monoclonal antibody preparations used to date have been murine in origin. The Fc region of such mouse antibodies is a very poor activator of human immune function. Technical advances, allowing the production of human/humanized monoclonals (see later) may render this therapeutic approach more attractive in the future. 

Figure 13.5 Outline of the production strategy of CEA-SCAN. The antibody-producing hybridoma cell line was originally obtained by standard methods of hybridoma generation. Spleen-derived murine B-lymphocytes were fused with murine myeloma calls. The resulting stable hybridomas were screened for the production of anti-CEA monoclonals. The clone chosen produces an IgG anti-CEA antibody. Note that the finished product outlined above is not radiolabelled. The freeze-dried antibody preparation (which has a shelf life of 2 years at 2-8 °C) is reconstituted immediately prior to its medical use. The reconstituting solution contains 99mTc, and is formulated to facilitate direct conjugation of the radiolabel to the antibody fragment...

Despite the scientific elegance of the antibody-mediated approach to tumour detection/destruc-tion, initial clinical trials proved disappointing. A number of factors contributed to their poor therapeutic performance, particularly against solid tumours. Most such factors relate directly/ indirectly to the fact that the first generation of such drugs utilized whole monoclonal antibody preparations of murine origin. These factors include ... 

Table 13.4 The serum half-life values of some IgG antibody preparations when administered to humans...

Various antibody preparations have been developed that facilitate imaging of vascular-related conditions, including myocardial infarction, deep vein thrombosis and atherosclerosis. Anti-myosin monoclonal antibody fragments (Fab) labelled with mIn, for example, have been used for imaging purposes in conjunction with a planar gamma camera. The antibody displays specificity for intracellular cardiac myosin, which is exposed only upon death of heart muscle tissue induced by a myocardial infarction (heart attack). 

The technique may also be done in a single stage if the initial antibody is labelled but this is less convenient and often more expensive than having a single labelled antibody preparation that can be used to detect all antibodies from a single species of donor. 

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

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