Monoclonal antibodies specificity testing

The first mouse monoclonal antibody specific for human CD3 was produced in 1979 and named orthoclone OKT3. Aside from its use in the laboratory, OKT3 became the first anti-CD3 antibody to be utilized in transplantation medicine, but its wider application was hampered by its immunogenic and mitogenic properties (reviewed in [6]). Consequently, humanized and engineered anti-CD3 antibodies were developed to circumvent these limitations (Table 1). Since T cells and the TCR are involved in many immunological diseases, it is not surprising that the application of CD3 antibodies is not restricted to the field of transplantation. For example, CD3 antibodies are tested in clinical studies of diseases such as autoimmune diabetes (type 1 diabetes), immune-mediated inflammatory arthritis and inflammatory bowel disease [7]. 

Figure 7.1. Protein expression mapping using an antibody array. The antibody array consists of monoclonal antibodies specific for a set of proteins in the organism of interest gridded onto a filter. To determine if a protein is expressed under the conditions being tested, a crude lysate is obtained and the proteins within the lysate are labeled with a fluorescent tag. The lysate is applied to the filter and the proteins are allowed to bind to the relevant antibody. Bound proteins are visualized via the fluorescent tag.

Dilute anti-(mouse-lgG)-lgG (in the case of monoclonal antibodies for testing conjugates prepared from antibodies of other origin, use the respective anti-species specific antibody) to 5 pg/ml in Soln. A. Coat the wells of a microtest plate with 100 pl/well of this dilution and incubate at 4 °C overnight. Remove antibody solution and wash once with TBS. Add 150 pi Soln. C per well and incubate at... 

This review is intended to provide helpful information regarding potential monoclonal antibody IgG test article (non-CDR-mediated) binding to specific tissues, cell types or other tissue elements, and subcellular locations following exogenous intravenous administration in animal model test systems. Particulars specific to other routes of administration were reviewed by Lobo et al. [1]. 

The development of monoclonal antibody probes specific to human albumin is the subject of this chapter. These antibody probes and the test methods were developed and evaluated for use in forensic science situations. Although these methods work well on soluble extracts of dried blood stains that are several years old, they have not been applied to archaeological material. The successful development of monoclonal antibodies specific to the human albumin molecule suggests that an approach similar to that used for identifying human tissues and blood on forensic evidence could be applied to any species of interest to the archaeologist. 

Several ELISA formats have been considered, and the one currently being tested is called the "hapten tracer" method. First introduced to this project by Dr. Freya Jung while a postdoctoral fellow in Dr. Hammock s laboratory, this method uses microtiter plates coated with a commercial preparation of goat anti-mouse antibody. Enzyme-labeled hapten competes with analyte for receptor sites on a mouse monoclonal antibody specific for the analyte. The amount of enzyme left after washing is inversely proportional to the... 

Supernatant (125 uL) was removed from the well for screening by EIA when the cells of the colony were one-quarter to one-half confluent. Cells from colonies testing positive for anti-picloram antibody activity were transfered to 24 well plates, rescreened by EIA and transfered again into 25 cm flasks if they remained positive for picloram antibodies. The limiting dilution procedure was repeated to ensure monoclonality. After a final assessment by EIA, the cells producing the monoclonal antibodies specific for picloram were collected for the production of ascitic fluid in mice. 

In a random set of 28 B.t. strains tested with monoclonal antibodies specific for type 3 toxin, only 8 showed positive reaction. In bioassay only the same 8 strains, and only these, exhibited toxicity to S. littoralis. Thus a perfect correlation exists between the antigenic structure of B.t. toxins and their insecticidal spectrum. 

A specific antibody is desirable because it enables an IA to be performed with limited or no sample clean-up. For reagent-excess methods, it is very important to choose specific antibodies. Cross-reactivity of polyclonal antiserum from each blood collection or each clone of monoclonal antibodies is tested against known metabolites, drug degradants, concomitant drugs, and the protein carrier... 

The stick test has been further modified using monoclonal antibodies specific for OA and CTX that are more specific than the sheep antibody (Hokama et al. 1986 Taizo 1987). These monoclonal antibodies are in the process of being evaluated. A preliminary collaborative evaluative study of the rapid enzyme immunoassay stick test has been concluded. Eight of the nine laboratories... 

Early clinical trials of antibody-targeted drug-loaded liposomes have already demonstrated some promising results. Thus, doxorubicin-loaded PEGylated liposomes (with the size of approx. 140 nm) modified with F(ab )2 fragments of the GAFI monoclonal antibody specific for the stomach cancer were tested in a Phase I clinical studies and demonstrated the pharmacokinetics similar to that of Doxil. ... 

Commercial use of cell and tissue culture continues to expand. Improvement of organisms through recombinant nucleic acid techniques has become commonplace. Formerly, a few laboratories were well ahead of most others, but now the methods have been perfected for routine use. Another technique that is widely practiced is culturing of cells that excrete high concentrations of just one antibody protein. The specificity of antibodies and antigens is exploited in medical testing procedures using these pure monoclonal antibodies. 

The limitations of ELISA methods include the specificity of antibodies, the concentrations of primary antibody and antigen, and the type of reaction solution. Nonspecific binding of either of the antibodies to related antigens, unrelated proteins of other bacteria, or even the microtiter plate may lead to false positive reactions.49,52 57 Use of a monoclonal antibody may decrease crossreactivity with other antigens. For detection of low numbers of bacteria, as in drinking water, the sample may be filtered to concentrate the cells or cultured in a selective broth until it reaches the minimum detection limit for ELISA.49,58 Commercial test kits using dipsticks, immunoblots, and sandwich ELISA methods have been developed for the identification of pathogenic bacteria.58,59... 

Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays that can be used to screen test samples routinely for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive. 

This approach appears somewhat irrational and without much scientific merit, since many of these new molecules are minimally toxic or nontoxic by this sort of acute evaluation. As in the case of interferons or monoclonal antibodies, the toxic effects observed in humans might not be predicted from safety assessments in rodents. An appropriate test species should be selected. Is the rat or mouse the appropriate species to evaluate a species-specific rDNA protein such as human growth hormone or interferons, or would nonhuman primates be more suitable Does the nonhuman primate really offer any advantages There is some consensus that the nonhuman primate may be a more appropriate species for testing some rDNA human proteins. 

Species differences must be considered when choosing a model and, in particular, species-specific immunological differences between the human and the test animal. For example, in humans, an anti-CD4 monoclonal antibody (MAb) will bind to CD4 expressed on monocytes, with subsequent fixing of complement and destruction of antigen-presenting cells. However, since CD4 molecules are not expressed on murine monocytes, these effects would not be evident in a murine model. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

Separation of a mixture of proteins by electrophoretic techniques such as polyacrylamide gel, SDS polyacrylamide or iso-electric focusing usually results in a complex pattern of protein bands or zones. Interpretation of the results often involves a comparison of the patterns of test and reference mixtures and identification of an individual protein, even using immunoelectrophoresis (Figure 11.15), is very difficult. However, specific proteins can often be identified using an immunoblotting technique known as Western blotting. The prerequisite is the availability of an antibody, either polyclonal or monoclonal, against the test protein. 

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