Antibodies properties, Table

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]. 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

There are 20 amino acids found in the human body that are involved in protein synthesis. Refer to your textbook for a table of the 20 amino acids. The sequence of the amino acids dictates the properties of a protein. Examples of proteins include keratin in hair, hemoglobin, insulin, antibodies, and enzymes. 

Although many individual biomolecular functions have already been studied by labeling bioactive molecules, proteins, antibodies and DNA strands with organic dyes and quantum dots, the extraordinary properties of silver clusters suggest that these species might be competitive alternatives (Table 1 and Fig. 11a). Nevertheless, silver clusters present some disadvantages in their application to biological systems. 

TABLE 10.2. Physiological properties and disposition of antibodies and serum albumin in humans... 

As part of SW-846, the EPA has validated and approved many immunoassay and colorimetric screening methods for a wide range of contaminants, such as petroleum fuels, pesticides, herbicides, PCBs, and explosives. Immunoassay technology uses the property of antibodies to bind to specific classes of environmental pollutants allowing fast and sensitive semiquantitative or qualitative detection. Colorimetric kits are based on the use of chemical reactions that indicate the presence of target analytes by a change in color. Table 3.9 presents a summary of EPA-approved screening methods and their detection capabilities. 

The method of choice is dependent upon the analyte, the assay performance required to meet the intended application, the timeline, and cost-effectiveness. The assay requirements include sensitivity, selectivity, linearity, accuracy, precision, and method robustness. Assay sensitivity in general is in the order of IA > LC-MS/MS > HPLC, while selectivity is IA LC-MS/MS > HPLC. However, IA is an indirect method which measures the binding action instead of relying directly on the physico-chemical properties of the analyte. The IA response versus concentration curve follows a curvilinear relationship, and the results are inherently less precise than for the other two methods with linear concentration-response relationships. The method development time for IA is usually longer than that for LC/MS-MS, mainly because of the time required for the production and characterization of unique antibody reagents. Combinatorial tests to optimize multiple factors in several steps of some IA formats are more complicated, and also result in a longer method refinement time. The nature of IAs versus that of LC-MS/MS methods are compared in Table 6.1. However, once established, IA methods are sensitive, consistent, and very cost-effective for the analysis of large volumes of samples. The more expensive FTMS or TOF-MS methods can be used to complement IA on selectivity confirmation. 

Alcohol oxidase could be immobilized onto DEAE-sepharose (Pharmacia) and had very high stability even when dried and rehydrated. We therefore tried to stabilize alcohol oxidize on a solid surface and as free enzyme using DEAE dextran. However this was not successful (Fig. 5). We also knew that lactitol had been used in stabilizing various antibodies during the manufacture of antibody based tests, however lactitol also had poor stabilizing properties when dried with alcohol oxidase (Fig 5). Unexpectedly, however, we found that a combination of DEAE dextran and lactitol gave excellent stabilization of die enzyme (Fig 5.). Further experiments showed that the combination of stabilizers was generic and would be used to stabilize broad selection of enzymes (Table 1 and see ref. 17). 

TABLE I Molecular Properties of Antibodies and Related Solid-Phase Separation Methods... 

Isoelectric precipitation and acid precipitation are also used to separate antibodies. Isoelectric precipitation (also called euglobulin precipitation) uses the solubility properties of a protein near its isoelectric point.69 When a concentrated protein solution in a low ionic strength buffer is titrated to its isoelectric point, it precipitates very slowly. Table 4 shows a balance of a process for purification of murine monoclonal IgM antibodies.70 The success of precipitation can be followed by the cumulative IgM content in the centrifugate and the sediment. The procedure is very gentle, but very sensitive to environmental conditions. 

In spite of the frequent use of ammonium sulfate, other hydrophobic association promoting salts can be useful at neutral pH. In that way, it is possible to select the degree of lyotropic property adapted to the antibody to purify (see Table 6). 

It was shown I8 that the binding process is the rate-limiting step, with an adsorption rate constant of kd = 4.8 x 104 dm mol s. The calculated mass transfer coefficient for the diffusion into the pores of the support contributes 11% to the overall adsorption process. The value of ka is an order of magnitude lower than that reported in Table 2. The binding properties of the polyclonal immunoadsorbents used in these two studies may differ because of the different methods employed for protein immobilization. Another possible explanation may be an underestimation of the contribution for the diffusion rate-limiting step as the polyclonal anti-HSA antibody was attached to a silica matrix of large pores [18]. 

Table 2.11. Properties conferred on human antibodies by constant regions...

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